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Expression of Bioactive Thymosin Alpha 1 (T?1) in Marker-free Transgenic Lettuce ( Lactuca sativa )
Thymosin ?1 (T?1) was widely used for the treatment of hepatitis (B and C) and several cancers. However, current production\\u000a of T?1 is difficultly meeting clinical needs. To develop a novel and safety approach for T?1 production, we synthesized a\\u000a T?1 gene (124Ābp) based on the plant codon usage bias and constructed a four-copy T?1 gene concatemer (408Ābp) by using
Lijie Cui; Yuhui Chen; Guoan Shen; Lingxia Zhao; Kexuan Tang
Immunopathogenic role of T H1 cells in autoimmune diabetes: Evidence from a T1 and T2 doubly transgenic non-obese diabetic mouse model
To improve the feasibility of in vivo monitoring of autoreactive T cells in the diabetogenic process, we generated T1 and T2 doubly transgenic non-obese diabetic (NOD) mice in which transgenic human CD90 (hCD90) is simultaneously expressed on IFN-?-producing cells or murine CD90.1 (mCD90.1) is expressed on IL-4-producing cells. These transgenic NOD mice develop diabetes with the same kinetics and incidence
Jung-Tung Hung; Jen-Hsiang Liao; Yu-Chung Lin; Hsiu-Ying Chang; Shu-Fen Wu; Tsung-Hsien Chang; John T. Kung; Shie-Liang Hsieh; Hugh McDevitt; Huey-Kang Sytwu
The resistance of transgenic papaya breeding lines to Papaya ringspot virus (PRSV) was examined. Resistance was conferred by non-translatable transgenes derived from the coat protein (CP) gene of a PRSV isolate (H1K) from Florida. To render the CP gene non-translatable, either a stop-codon (D6 lines) or frame-shift (X17-2 lines) mutation had been intro- duced into the CP gene. Non-transgenic and
[Obtaining the transgenic lines of finger millet Eleusine coracana (L.) Gaertn. With dinitroaniline resistance].
The current data is dedicated to the study of bioballistic and Agrobacterium-mediated transformation of finger millet with the constructs carrying the mutant alpha-tubulin gene (TUAm 1), isolated from R-biotype goosegrass (Eleusine indica L.), for the decision of problem of dinitroaniline-resistance. It was found that 10 microM of trifluralin is optimal for the selection of transgene plants of finger millet. PCR analysis of transformed lines confirmed the transgene nature of plants. The analysis of seed of T1 oftransgene lines confirmed heterozygous character of inheritance of the resistance. PMID:25016822
Transgene integration and chromosome alterations in two transgenic lines of tritordeum
Plants from two transgenic lines of tritordeum (an amphiploid between Triticum turgidum cv. durum and Hordeum chilense) have been analyzed by fluorescence in-situ hybridization (FISH) to characterize the transgene integration sites and chromosome rearrangements. Transgenic lines were\\u000a transformed in two different events with the genes encoding for the high-molecular-weight glutenin subunits (HMW-GS), 1Ax1\\u000a and\\/or 1Dx5. Three integration sites and four
F. Barro; A. MartĒn; A. Cabrera
Skin Fibroblasts from Patients with Type 1 Diabetes (T1D) Can Be Chemically Transdifferentiated into Insulin-Expressing Clusters: A Transgene-Free Approach
The conversion of differentiated cells into insulin-producing cells is a promising approach for the autologous replacement of pancreatic cells in patients with type 1 diabetes (T1D). At present, cellular reprogramming strategies encompass ethical problems, epigenetic failure or teratoma formation, which has prompted the development of new approaches. Here, we report a novel technique for the conversion of skin fibroblasts from T1D patients into insulin-expressing clusters using only drug-based induction. Our results demonstrate that skin fibroblasts from diabetic patients have pancreatic differentiation capacities and avoid the necessity of using transgenic strategies, stem cell sources or global demethylation steps. These findings open new possibilities for studying diabetes mechanisms, drug screenings and ultimately autologous transgenic-free regenerative medicine therapies in patients with T1D.
Pereyra-Bonnet, Federico; Gimeno, Maria L.; Argumedo, Nelson R.; Ielpi, Marcelo; Cardozo, Johana A.; Gimenez, Carla A.; Hyon, Sung-Ho; Balzaretti, Marta; Loresi, Monica; Fainstein-Day, Patricia; Litwak, Leon E.; Argibay, Pablo F.
Response of sensitive human ataxia and resistant T-1 cell lines to accelerated heavy ions
The radiation dose responses of fibroblast from a patient with Ataxia telangiectasis (AT-2SF) and an established line of human T-1 cells were studied. Nearly monoenergetic accelerated neon and argon ions were used at the Berkeley Bevalac with various residual range values. The LET of the particles varied from 30 keV/..mu..m to over 1000 keV/..mu..m. All Ataxia survival curves were exponential functions of the dose. Their radiosensitivity reached peak values at 100 to 200 keV/..mu..m. Human T-1 cells have effective sublethal damage repair as has been evidenced by split dose experiments, and they are much more resistant to low LET than to high LET radiation. The repair-misrepair model has been used to interpret these results. We have obtained mathematical expressions that describe the cross sections and inactivation coefficients for both human cell lines as a function of the LET and the type of particle used. The results suggest either that high-LET particles induce a greater number of radiolesions per track or that heavy-ions at high LET induce lesions that kill cells more effectively and that are different from those produced at low LET. We assume that the lesions induced in T-1 and Ataxia cells are qualitatively similar and that each cell line attempts to repair these lesions. The result in most irradiated Ataxia cells, however, is either lethal misrepair or incomplete repair leading to cell death. 63 references, 10 figures, 1 table.
Tobias, C.A.; Blakely, E.A.; Chang, P.Y.; Lommel, L.; Roots, R.
Establishment and characterization of CAG\\/EGFP transgenic rabbit line
Cell marking is a very important procedure for identifying donor cells after cell and\\/or organ transplantation in vivo. Transgenic\\u000a animals expressing marker proteins such as enhanced green fluorescent protein (EGFP) in their tissues are a powerful tool\\u000a for research in fields of tissue engineering and regenerative medicine. The purpose of this study was to establish transgenic\\u000a rabbit lines that ubiquitously
Ri-ichi Takahashi; Takashi Kuramochi; Kazuki Aoyagi; Shu Hashimoto; Ichiro Miyoshi; Noriyuki Kasai; Yoji Hakamata; Eiji Kobayashi; Masatsugu Ueda
Accumulation of nickel in transgenic tobacco
NASA Astrophysics Data System (ADS)
The accumulation of heavy metal Ni in the roots and leaves of four T1 transgenic lines of tobacco (T(1)20E, T(1)24C, T(1)18B1 and T(1)20B) expressing eiMT1 from E.indica was assessed. The aim of the study was to investigate the level of Ni accumulation in the leaves and roots of each transgenic lines and to evaluate the eligibility of the plants to be classified as a phytoremediation agent. All of the transgenic lines showed different ability in accumulating different metals and has translocation factor (TF) less than 1 (TF<1) at all levels of metal treatment. Among the 4 transgenic lines, transgenic line T(1)24C showed the highest accumulation of Ni (251.9 ▒ 0.014 mg/kg) and the lowest TF value (TFT(1)24C=0.0875) at 60 ppm Ni.
Sidik, Nik Marzuki; Othman, Noor Farhan
A selectively terminable transgenic rice line expressing human lactoferrin.
Human lactoferrin (hLF) is a multifunctional milk protein which could be utilized for promoting human health. Transgenic rice has been used as a bioreactor for mass production of recombinant hLF. However, one major concern over such transgenic rice is the risk of its unintended spreading into environment and into our food supplies. Here we report the development of selectively terminable transgenic rice expressing human lactoferrin in seeds. These transgenic rice plants could be selectively terminated by bentazon, a common herbicide used for rice weed control. The hLF expression cassette was constructed into a T-DNA containing the RNA interference cassette suppressing the expression of the rice gene CYP81A6 which detoxifies herbicide bentazon, and the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) cassette which confers to glyphosate tolerance. A transgenic line, named as G281, was identified for its high sensitivity to bentazon, high tolerance to glyphosate, and high expression of hLF. Southern analysis suggested G281 is a single copy insertion event. Field tests demonstrated that G281 plants can be completely killed by a single spray of bentazon at 1000 mg/L, which is safe to regular rice and represents only half of the dose recommended by manufacturer for rice field weed control. Therefore, any G281 contaminations in regular rice could be selectively terminated to make sure it will not enter food or feed supplies. PMID:20433928
Lin, Chaoyang; Nie, Peng; Lu, Wei; Zhang, Qing; Li, Jing; Shen, Zhicheng
Transgene-specific and event-specific molecular markers for characterization of transgenic papaya lines resistant to Papaya ringspot virus.
The commercially valuable transgenic papaya lines carrying the coat protein (CP) gene of Papaya ringspot virus (PRSV) and conferring virus resistance have been developed in Hawaii and Taiwan in the past decade. Prompt and sensitive protocols for transgene-specific and event-specific detections are essential for traceability of these lines to fulfill regulatory requirement in EU and some Asian countries. Here, based on polymerase chain reaction (PCR) approaches, we demonstrated different detection protocols for characterization of PRSV CP-transgenic papaya lines. Transgene-specific products were amplified using different specific primer pairs targeting the sequences of the promoter, the terminator, the selection marker, and the transgene, and the region across the promoter and transgene. Moreover, after cloning and sequencing the DNA fragments amplified by adaptor ligation-PCR, the junctions between plant genomic DNA and the T-DNA insert were elucidated. The event-specific method targeting the flanking sequences and the transgene was developed for identification of a specific transgenic line. The PCR patterns using primers designed from the left or the right flanking DNA sequence of the transgene insert in three selected transgenic papaya lines were specific and reproducible. Our results also verified that PRSV CP transgene is integrated into transgenic papaya genome in different loci. The copy number of inserted T-DNA was further confirmed by real-time PCR. The event-specific molecular markers developed in this investigation are crucial for regulatory requirement in some countries and intellectual protection. Also, these markers are helpful for prompt screening of a homozygote-transgenic progeny in the breeding program. PMID:19526355
Fan, Ming-Jen; Chen, Shu; Kung, Yi-Jung; Cheng, Ying-Huey; Bau, Huey-Jiunn; Su, Tien-Tsai; Yeh, Shyi-Dong
Inheritance of transgenes in transgenic Bt lines resistance to Helicoerpa armigera in upland cotton.
Six transgenic Bt cotton cultivars (lines) including GKsu12, GK19, MR1, GK5, 109B, and SGK1 are highly resistant to bollworm from the seedling to boll-setting stages in bioassays with detached cotton leaves, though there are differences in resistant level and Bt toxin content in these transgenic cottons. Genetics analysis reveals that the resistance to Helicoverpa armigera in these six transgenic Bt cotton cultivars (lines) are controlled by one pair of dominant genes. Allelic tests further demonstrate some populations are in Mendel segregation for two nonallelic genes, i.e., the inserted Bt gene in GKsu12 is nonallelic to that of SGK1, GK5, 109B, and GK19 and Bt genes in GK19 and SGK1 are likely inserted in the same or in close proximity (genetically closely linked), while some F(2) produce abnormal segregation patterns, with a segregation of resistance to Helicoerpa armigera vary between 15:1 and 3:1, though their Bt segregation fit into 15:1 by PCR analysis, suggesting Bt gene silence in these populations. Two genes silence may occur in these populations due to the homologous sequence by crossing since the silenced individuals accounted for 1/16 of the F(2) populations for allelic test. To those silenced populations, one of their parents all showed high resistance to bollworm. PMID:23143492
Zhang, Baolong; Guo, Wangzhen; Zhang, Tianzhen
Generation of Doubled Haploid Transgenic Wheat Lines by Microspore Transformation
Microspores can be induced to develop homozygous doubled haploid plants in a single generation. In the present experiments androgenic microspores of wheat have been genetically transformed and developed into mature homozygous transgenic plants. Two different transformation techniques were investigated, one employing electroporation and the other co-cultivation with Agrobacterium tumefaciens. Different tissue culture and transfection conditions were tested on nine different wheat cultivars using four different constructs. A total of 19 fertile transformants in five genotypes from four market classes of common wheat were recovered by the two procedures. PCR followed by DNA sequencing of the products, Southern blot analyses and bio/histo-chemical and histological assays of the recombinant enzymes confirmed the presence of the transgenes in the T0 transformants and their stable inheritance in homozygous T1?2 doubled haploid progenies. Several decisive factors determining the transformation and regeneration efficiency with the two procedures were determined: (i) pretreatment of immature spikes with CuSO4 solution (500 mg/L) at 4░C for 10 days; (ii) electroporation of plasmid DNA in enlarged microspores by a single pulse of ?375 V; (iii) induction of microspores after transfection at 28░C in NPB-99 medium and regeneration at 26░C in MMS5 medium; (iv) co-cultivation with Agrobacterium AGL-1 cells for transfer of plasmid T-DNA into microspores at day 0 for <24 hours; and (v) elimination of AGL-1 cells after co-cultivation with timentin (200¢400 mg/L).
Liu, Weiguo; Konzak, Calvin F.; von Wettstein, Diter; Rustgi, Sachin
Decoding method for T1 line format for ccitt 32K bit per second adpcm clear channel transmission and 64 KBPS clear channel transmission
In a data transmission system having first and second digital switching systems connected via T1 line transmission facilities for bidirectional data transmissions, a decoding method for T1 line zero bit suppression is described comprising the steps of: receiving an encoded T1 line frame including bundled channels; testing indicator bits of the received T1 line frame to determine whether any channel of the T1 line frame has been altered for the transmission; first transferring the received T1 line frame as received, performed in response to the indicated bits showing that no alteration have been made to the received T1 line frame; decoding mapping bits of the received T1 line frame to determine which channels of the bundle of the received T1 line frame have been altered for the encoded transmission; replacing the contents of each of the altered channel of the bundle with zeroes; iterating the steps of decoding and replacing for each of the bundles of the received T1 line frame; and second transferring the received T1 line frame with the altered channels being replaced with zeroes.
Blondeau, E.E. Jr.; Czarnecki, S.J.
Transgene-specific and event-specific molecular markers for characterization of transgenic papaya lines resistant to Papaya ringspot virus
The commercially valuable transgenic papaya lines carrying the coat protein (CP) gene of Papaya ringspot virus (PRSV) and conferring virus resistance have been developed in Hawaii and Taiwan in the past decade. Prompt and sensitive\\u000a protocols for transgene-specific and event-specific detections are essential for traceability of these lines to fulfill regulatory\\u000a requirement in EU and some Asian countries. Here, based
Ming-Jen Fan; Shu Chen; Yi-Jung Kung; Ying-Huey Cheng; Huey-Jiunn Bau; Tien-Tsai Su; Shyi-Dong Yeh
Efficient selection and evaluation of transgenic lines of Crambe abyssinica
Crambe abyssinica is a dedicated oilseed crop suitable for production of industrial feedstocks. Genetic modification of crambe has progressed substantially in the last few years, but the transformation efficiency needs to be further improved. Meanwhile, developing a reliable molecular system including Southern blot and qRT-PCR analyses is desired for effectively evaluating transgenic lines and gene expression levels of both endogenous and transgenes. In this study, we have developed an efficient transformation protocol with hygromycin as the selective agent for crambe transformation. In the regeneration test, addition of hygromycin at concentration of 5 mg L?1 resulted in 18% of shoot regeneration using crambe hypocotyls as explants, while no regeneration occurred when the hygromycin concentration reached 10 mg L?1. Based on this result, the hygromycin concentration up to 10 mg L?1 was used in the subsequent transformations. The results showed that the transformation efficiency under constant low selection pressure (H3-H3) was similar to that under higher selection pressure first, followed by transfer to lower selection pressure (H10-H3). The PCR, Southern blot and fatty acid composition analyses confirmed the integration of transgenes in the crambe genome. We have also optimized the Southern and qRT-PCR methods for future studies on crambe or related species. For Southern blot analysis on crambe, more than 50 ?g DNA is required for a clear band. The choice of enzymes for DNA digestion was not rigid for confirmation of the T-DNA integration, while for determining the copy number of transgenes, suitable enzymes should be chosen. Increasing the enzyme concentration could improve the digestion and 20 ?l enzyme was recomended for a complete digestion of up to 80 ?g crambe DNA. For qRT-PCR analysis, around 20 days after flowering was observed to be the suitable sampling time for expresseion analysis of genes invovled in the seed oil biosynthesis.
Li, Xueyuan; Fan, Jing; Gruber, Jens; Guan, Rui; Frentzen, Margrit; Zhu, Li-Hua
In vivo immunomodulatory effects of Antrodia camphorata polysaccharides in a T1/T2 doubly transgenic mouse model for inhibiting infection of Schistosoma mansoni
Antrodia camphorata (A. camphorata) is a fungus commonly used for treatment of viral hepatitis and cancer in Chinese folk medicine. Extract of A. camphorate is reported to possess anti-inflammatory, antihepatitis B virus and anticancer activities. In this study, we tested the in vivo effects of polysaccharides derived from A. camphorata (AC-PS) on immune function by detection of cytokine expression and evaluation of the immune phenotype in a T1/T2 doubly transgenic mouse model. The protective effect of AC-PS in mice was tested by infection with Schistosoma mansoni. The induction of large amounts of IFN-{gamma}, IL-2 and TNF-a mRNA were detected after 2 and 4 weeks of oral AC-PS administration in BALB/c and C57BL/6 mice. In transgenic mice, 3 to 6 weeks of oral AC-PS administration increased the proportion of CD4{sup +} T cells and B cells within the spleen. More specifically, there was an increase of Th1 CD4{sup +} T cells and Be1 cells among spleen cells as observed by detection the of Type1/Type2 marker molecules. By using a disease model of parasitic infection, we found that AC-PS treatment inhibited infection with S. mansoni in BALB/C and C57BL/6 mice. AC-PS appears to influence the immune system of mice into developing Th1 responses and have potential for preventing infection with S. mansoni.
Cheng, P.-C. [Institute of Tropical Medicine, National Yang-Ming University, Taipei, Taiwan (China); Hsu, C.-Y. [Institute of Molecular and Cellular Biology, National Tsing-Hua University, Hsinchu, Taiwan (China); Chen, C.-C. [Biotechnology Center, Grape King Inc., Chungli, Taiwan (China); Lee, K.-M. [Institute of Tropical Medicine, National Yang-Ming University, Taipei, Taiwan (China); Institute of Medical Biotechnology, Central Taiwan University of Science and Technology, Taichung, Taiwan (China)], E-mail: email@example.com
Human cyclin T1 expression ameliorates a T-cell-specific transcriptional limitation for HIV in transgenic rats, but is not sufficient for a spreading infection of prototypic R5 HIV1 strains ex vivo
BACKGROUND: Cells derived from native rodents have limits at distinct steps of HIV replication. Rat primary CD4 T-cells, but not macrophages, display a profound transcriptional deficit that is ameliorated by transient trans-complementation with the human Tat-interacting protein Cyclin T1 (hCycT1). RESULTS: Here, we generated transgenic rats that selectively express hCycT1 in CD4 T-cells and macrophages. hCycT1 expression in rat T-cells
Nico Michel; Christine Goffinet; Kerstin Ganter; Ina Allespach; Vineet N KewalRamani; Mohammed Saifuddin; Dan R Littman; Warner C Greene; Mark A Goldsmith; Oliver T Keppler
Metal mutagenesis in transgenic Chinese hamster cell lines.
Metals are toxic agents for which genotoxic effects are often difficult to demonstrate. To study metal mutagenesis, we have used two stable hprt/gpt+ transgenic cell lines that were derived from Chinese hamster V79 cells. Both the G12 and G10 cell lines are known to be very sensitive to clastogens such as X-rays and bleomycin, with the mutagenic response of the integrated xanthine guanine phosphoribosyl transferase (gpt) gene in G10 usually exceeding that of the same gene in the transgenic G12 cells. In studies with carcinogenic insoluble nickel compounds, a high level of mutagenesis was found at the gpt locus of G12 cells but not at the endogenous hypoxanthine phosphoribosyl transferase (hprt) locus of V79 cells. We have since demonstrated the similar recovery of a high frequency of viable G12 mutants with other insoluble nickel salts including nickel oxides (black and green). The relative mutant yield for the insoluble nickel compounds (G12 > G10) is the opposite of that obtained with nonmetal clastogens (G10 > G12). In the G12 cells, nickel mutagenesis may be related to the integration of the gpt sequence into a heterochromatic region of the genome. For some of the insoluble nickel compounds, significant inhibition of both cytotoxicity and mutant yield resulted when the G12 cells were pretreated with vitamin E. In comparison with the nickel studies, the mutagenic responses to chromium compounds in these cell lines were not as dramatic. Mutagenesis of the gpt target could not be demonstrated with other metals such as mercury or vanadium.
Klein, C B; Kargacin, B; Su, L; Cosentino, S; Snow, E T; Costa, M
Cadmium and zinc induction of ZnT-1 mRNA in an established carp cell line.
The cDNA of a zinc transporter-1 (ZnT-1) gene was cloned from an established cell line derived from common carp (Cyprinus carpio) using rapid amplification of cDNA ends (RACE). Using real-time quantitative PCR, we showed that both zinc (Zn) and cadmium (Cd) transiently upregulate ZnT-1 mRNA to comparable levels. The loosely bound cellular Zn pool, as estimated using the Zn-specific probe FluoZin-3, was increased threefold after exposure to 250 microM ZnCl(2). Correspondingly, the ZnT-1 mRNA level at 24 h was induced about fivefold, reflecting the need for more zinc export capacity. Total cellular zinc levels were not different from the controls after 72 h of exposure to 10, 50, or 250 microM ZnCl(2). A loss of total cellular Zn but little labile zinc changes were observed with up to 25 microM Cd. At 72 h, the total Zn was partially restored to the control levels, only 1 microM Cd allowed for a full recovery. Downregulation of ZnT-1 mRNA and partial loss of loosely bound Zn were observed with 50 microM Cd. Our results clearly show that although Zn and Cd can both regulate Zn export in EPC cells, the effects on the cellular Zn pools are quite different. PMID:16627006
Muylle, Frederik; Robbens, Johan; De Coen, Wim; Timmermans, Jean-Pierre; Blust, Ronny
Stability of transgene expression, field performance and recombination breeding of transformed barley lines
Stable inheritance of the transgene, consistent expression and competitive agronomic properties of transgenic crops are important\\u000a parameters for successful use of the latter. These properties have been analyzed with 18 homozygous transgenic barley lines\\u000a of the cultivar Golden Promise. The lines originated from three independent primary transformants obtained by the biolistic\\u000a method with three plasmids containing respectively, the bar gene,
H. Horvath; L. G. Jensen; O. T. Wong; E. Kohl; S. E. Ullrich; J. Cochran; C. G. Kannangara; D. von Wettstein
Safety assessment of transgenic Bacillus thuringiensis rice T1c-19 in Sprague-Dawley rats from metabonomics and bacterial profile perspectives.
Bacillus thuringiensis rice is facing commercialization as the main food source in the near future. The unintended effects of genetically modified (GM) organisms are the most important barriers to their promotion. We aimed to establish a new in vivo evaluation model for genetically modified foods by using metabonomics and bacterial profile approaches. T1c-19 rice flour or its transgenic parent MH63 was used at 70% wt/wt to produce diets that were fed to rats for ? 90 days. Urine metabolite changes were detected using (1)H NMR. Denaturing gradient gel electrophoresis and real-time polymerase chain reaction (RT-PCR) were used to detect the bacterial profiles between the two groups. The metabonomics was analyzed for metabolite changes in rat urine, when compared with the non-GM rice group, where rats were fed a GM rice diet. Several metabolites correlated with rat age and sex but not with GM rice diet. Significant biological differences were not identified between the GM rice diet and the non-GM rice diet. The bacteria related to rat urine metabolites were also discussed. The results from metabonomics and bacterial profile analyses were comparable with the results attained using the traditional method. Because metabonomics and bacterial profiling offer noninvasive, dynamic approaches for monitoring food safety, they provide a novel process for assessing the safety of GM foods. PMID:22215564
Cao, Sishuo; He, Xiaoyun; Xu, Wentao; Luo, YunBo; Yuan, Yanfang; Liu, Pengfei; Cao, Bo; Shi, Hui; Huang, Kunlun
Line 63-1: A New Virus-resistant Transgenic Papaya
The disease resistance of a transgenic line expressing the coat protein (CP) gene of the mild strain of the papaya ringspot virus (PRSV) from Hawaii was further analyzed against PRSV isolates from Hawaii and other geographical regions. Line 63-1 originated from the same transformation experiment that resulted in line 55-1 from which the transgenic commercial cultivars, `Rainbow' and `SunUp', were
P. Tennant; M. T. Souza; M. M. Fitch; R. Manshardt; J. L. Slightom; D. Gonsalves
Accelerated growth performance and stable germ-line transmission in androgenetically derived homozygous transgenic mud loach, Misgurnus mizolepis
Three stable lines of fast growing isogenic homozygous transgenic mud loach M.mizolepis have been generated by inducing interspecific androgenesis between heterozygous transgenic mud loach sperm and UV-irradiated common carp (Cyprinuscarpio) eggs. The inseminated eggs were heat-shocked to restore diploidy and generate viable androgenetic homozygous transgenics. Homozygosity of the transgenic locus of the induced transgenic mud loach was evidenced by Southern
Yoon Kwon Nam; Young Sun Cho; Hyo Jong Cho; Dong Soo Kim
Ongoing murine T1 or T2 immune responses to the hepatitis B surface antigen are excluded from the liver that expresses transgene-encoded hepatitis B surface antigen.
Different protein- or DNA-based vaccination techniques are available that prime potent humoral and cellular, T1 or T2 immune responses to the hepatitis B surface Ag (HBsAg) in mice. T1 and T2 are immune responses with isotype profile indicating Th1 and Th2 immunoregulation. We tested whether HBsAg-specific immune responses can be established in transgenic mice that express HBsAg in the liver (HBs-tg mice) using either these different vaccination techniques or an adoptive transfer system. HBsAg-specific responses could not be primed in HBs-tg mice with the established, potent vaccine delivery techniques. In contrast, adoptive transfers of T1- and T2-type HBsAg-immune spleen cells into congenic HBs-tg hosts (that were not conditioned by pretreatment) suppressed HBsAg antigenemia and gave rise to HBsAg-specific serum Ab titers. The establishment of continuously rising anti-HBsAg serum Ab levels with alternative isotype profiles (reflecting T1 or T2 polarization) in transplanted HBs-tg hosts required donor CD4+ T cell-dependent restimulation of adoptively transferred immune cells by transgene-derived HBsAg. Injections of HBsAg-specific Abs into HBs-tg mice did not establish stable humoral immunity. The expanding T1 or T2 immune responses to HBsAg in HBs-tg hosts did not suppress transgene-directed HBsAg expression in the liver and did not induce liver injury. In addition to priming functional antiviral effector cells, the conditioning of the liver microenvironment to enable delivery of antiviral effector functions to this organ are therefore critical for effective antiviral defense. A major challenge in the development of a therapeutic vaccine against chronic hepatitis B or C virus infection is thus the efficient targeting of specifically induced immune effector specificities to the liver. PMID:10754320
Schirmbeck, R; Wild, J; Stober, D; Blum, H E; Chisari, F V; Geissler, M; Reimann, J
Rapid Production of Multiple Independent Lines of Fertile Transgenic Wheat (Triticum aestivum).
Improvement of wheat (Triticum aestivum) by biotechnological approaches is currently limited by a lack of efficient and reliable transformation methodology. In this report, we detail a protocol for transformation of a highly embryogenic wheat cultivar, Bobwhite. Calli derived from immature embryos, 0.5 to 1 mm long, were bombarded with microprojectiles coated with DNA containing as marker genes the bar gene, encoding phosphinothricin-resistance, and the gene encoding [beta]-glucuronidase (GUS), each under control of a maize ubiquitin promoter. The bombardment was performed 5 d after embryo excision, just after initiation of callus proliferation. The ability of plantlets to root in the presence of 1 or 3 mg/L of bialaphos was the most reliable selection criteria used to identify transformed plants. Stable transformation was confirmed by marker gene expression assays and the presence of the bar sequences in high molecular weight chromosomal DNA of the resultant plants. Nine independent lines of fertile transgenic wheat plants have been obtained thus far, at a frequency of 1 to 2 per 1000 embryos bombarded. On average, 168 d elapsed between embryo excision for bombardment and anthesis of the T0 plants. The transmission of both the resistance phenotype and bar DNA to the T1 generation verified that germline transformation had occurred.
Weeks, J. T.; Anderson, O. D.; Blechl, A. E.
Tobacco Transgenic Lines That Express Fenugreek Galactomannan Galactosyltransferase Constitutively Have Structurally Altered Galactomannans in Their Seed Endosperm Cell Walls1
Galactomannans [(1?6)-?-d-galactose (Gal)-substituted (1?4)-?-d-mannans] are major cell wall storage polysaccharides in the endosperms of some seeds, notably the legumes. Their biosynthesis in developing legume seeds involves the functional interaction of two membrane-bound glycosyltransferases, mannan synthase (MS) and galactomannan galactosyltransferase (GMGT). MS catalyzes the elongation of the mannan backbone, whereas GMGT action determines the distribution and amount of Gal substitution. Fenugreek (Trigonella foenum-graecum) forms a galactomannan with a very high degree of Gal substitution (Man/Gal = 1.1), and its GMGT has been characterized. We now report that the endosperm cell walls of the tobacco (Nicotiana tabacum) seed are rich in a galactomannan with a very low degree of Gal substitution (Man/Gal about 20) and that its depositional time course is closely correlated with membrane-bound MS and GMGT activities. Furthermore, we demonstrate that seeds from transgenic tobacco lines that express fenugreek GMGT constitutively in membrane-bound form have endosperm galactomannans with increased average degrees of Gal substitution (Man/Gal about 10 in T1 generation seeds and about 7.5 in T2 generation seeds). Membrane-bound enzyme systems from transgenic seed endosperms form galactomannans in vitro that are more highly Gal substituted than those formed by controls under identical conditions. To our knowledge, this is the first report of structural manipulation of a plant cell wall polysaccharide in transgenic plants via a biosynthetic membrane-bound glycosyltransferase.
Reid, J.S. Grant; Edwards, Mary E.; Dickson, Cathryn A.; Scott, Catherine; Gidley, Michael J.
Novel cell lines derived from transgenic mice expressing recombinant human proteins
We have used transgenic mouse technology to establish immortalized hepatoma cell lines stably secreting heterologous proteins,\\u000a such as human ?1-antitrypsin and human factor IX. Hepatocyte-specific regulatory DNA sequences were used to target both the expression of\\u000a anonc gene and the gene coding for the human protein to the liver of transgenic mice which eventually developed hepatocellular\\u000a carcinomas. Tumour cells were
Frķdķric Perraud; Wilfried Dalemans; Dalila Ali-Hadji; Andrea Pavirani
Molecular analysis and quantitative detection of a transgenic rice line expressing a bifunctional fusion TPSP
The remarkable amount of public and scientific debate with regard to the safety of genetically modified (GM) crops has resulted in the implementation of mandatory risk assessments of newly developed GM crops, as well as mandatory labeling of GM crops and foods following their commercial release. Among the many currently available GM rice lines, transgenic rice lines that overexpress a
Soon-Chun Jeong; In Soon Pack; Eun-Young Cho; Eun Soo Youk; Sangkyu Park; Won Kee Yoon; Chang-Gi Kim; Yang Do Choi; Ju-Kon Kim; Hwan Mook Kim
Efficient screening of transgenic plant lines for ecological research.
Plants stably transformed to manipulate the expression of genes mediating ecological performance have profoundly altered research in plant ecology. Agrobacterium-mediated transformation remains the most effective method of creating plants harbouring a limited number of transgene integrations of low complexity. For ecological/physiological research, the following requirements must be met: (i) the regenerated plants should have the same ploidy level as the corresponding wild-type plant and (ii) contain a single transgene copy in a homozygous state; (iii) the T-DNA must be completely inserted without vector backbone sequence and all its elements functional; and (iv) the integration should not change the phenotype of the plant by interrupting chromosomal genes or by mutations occurring during the regeneration procedure. The screening process to obtain transformed plants that meet the above criteria is costly and time-consuming, and an optimized screening procedure is presented. We developed a flow chart that optimizes the screening process to efficiently select transformed plants for ecological research. It consists of segregational analyses, which select transgenic T? and T? generation plants with single T-DNA copies that are homozygous. Indispensable molecular genetic tests (flow cytometry, diagnostic PCRs and Southern blotting) are performed at the earliest and most effective times in the screening process. qPCR to quantify changes in transcript accumulation to confirm gene silencing or overexpression is the last step in the selection process. Because we routinely transform the wild tobacco, Nicotiana attenuata, with constructs that silence or ectopically overexpress ecologically relevant genes, the proposed protocol is supported by examples from this system. PMID:21518300
Gase, Klaus; Weinhold, Arne; Bozorov, Tohir; Schuck, Stefan; Baldwin, Ian T
Germline transmission of a novel rat embryonic stem cell line derived from transgenic rats.
Germline-competent rat embryonic stem (ES) cell lines are important resources for the creation of mutant rat models using ES-cell-based gene targeting technology. The ability to isolate germline-competent ES cell lines from any rat strain, including genetically modified strains, would allow for more sophisticated genetic manipulations without extensive breeding. Sprague Dawley (SD) males carrying an enhanced green fluorescent protein (EGFP) transgene were used as the founder animals for the derivation of ES cell lines. A number of ES cell lines were established and subjected to rigorous quality control testing that included assessment of pluripotency factor expression, karyotype analysis, and pathogen/sterility testing. Two male ES cell lines, SD-Tg.EC1/Rrrc and SD-Tg.EC8/Rrrc, were injected into blastocysts recovered from a cross of Dark Agouti (DA) males with SD females. Resulting chimeric animals were bred with wild-type SD mates to verify the germline transmissibility of the ES cell lines by identifying pups carrying the ES cell line-derived EGFP transgene. While both ES cell lines gave rise to chimeric animals, only SD-Tg.EC1 was germline competent. This confirms the feasibility of deriving germline-competent ES cell lines from transgenic rat strains and provides a novel ES cell line with a stable green fluorescent protein (GFP) reporter for future genetic manipulations to create new rat models. PMID:22455749
Men, Hongsheng; Bauer, Beth A; Bryda, Elizabeth C
Derivation of a germline competent transgenic Fischer 344 embryonic stem cell line.
Embryonic stem (ES) cell-based gene manipulation is an effective method for the generation of mutant animal models in mice and rats. Availability of germline-competent ES cell lines from inbred rat strains would allow for creation of new genetically modified models in the desired genetic background. Fischer344 (F344) males carrying an enhanced green fluorescence protein (EGFP) transgene were used as the founder animals for the derivation of ES cell lines. After establishment of ES cell lines, rigorous quality control testing that included assessment of pluripotency factor expression, karyotype analysis, and pathogen/sterility testing was conducted in selected ES cell lines. One male ES cell line, F344-Tg.EC4011, was further evaluated for germline competence by injection into Dark Agouti (DA) X Sprague Dawley (SD) blastocysts. Resulting chimeric animals were bred with wild-type SD mates and germline transmissibility of the ES cell line was confirmed by identification of pups carrying the ES cell line-derived EGFP transgene. This is the first report of a germline competent F344 ES cell line. The availability of a new germline competent ES cell line with a stable fluorescence reporter from an inbred transgenic rat strain provides an important new resource for genetic manipulations to create new rat models. PMID:23437152
Men, Hongsheng; Bryda, Elizabeth C
Efficient germ-line transmission obtained with transgene-free induced pluripotent stem cells.
Induced pluripotent stem (iPS) cells hold great promise for regenerative medicine. To overcome potential problems associated with transgene insertions, efforts have been directed over the past several years to generate transgene-free iPS cells by using non-viral-vector approaches. To date, however, cells generated through such procedures have had problems producing reproductively competent animals, suggesting that their quality needed further improvement. Here we report the use of optimized assemblies of reprogramming factors and selection markers incorporated into single plasmids as nonintegrating episomes to generate germ-line-competent iPS cells. In particular, the pMaster12 episome can produce transgene-free iPS cells that, when grown in 2i medium, recapitulate good mouse ES cells, in terms of their competency for generating germ-line chimeras. PMID:25002522
Wu, Sen; Wu, Yuanyuan; Zhang, Xi; Capecchi, Mario R
Transgenic Pm3b wheat lines show resistance to powdery mildew in the field.
Plant resistance (R) genes are highly effective in protecting plants against diseases, but pathogens can overcome such genes relatively easily by adaptation. Consequently, in many cases R genes do not confer durable resistance in agricultural environments. One possible strategy to make the use of R genes more sustainable depends on the modification of R genes followed by transformation. To test a possible transgenic use of R genes, we overexpressed in wheat the Pm3b resistance gene against powdery mildew under control of the maize ubiquitin promoter. Four independent transgenic lines were tested in the greenhouse and the field during 3?years. The four lines showed a five- to 600-fold transgene overexpression compared with the expression of the endogenous Pm3b gene in the landrace 'Chul'. Powdery mildew resistance was significantly improved in all lines in the greenhouse and the field, both with naturally occurring infection or after artificial inoculation. Under controlled environmental conditions, the line with the strongest overexpression of the Pm3b gene showed a dramatic increase in resistance to powdery mildew isolates that are virulent on the endogenous Pm3b. Under a variety of field conditions, but never in the greenhouse, three of the four transgenic lines showed pleiotropic effects on spike and leaf morphology. The highest overexpressing line had the strongest side effects, suggesting a correlation between expression level and phenotypic changes. These results demonstrate that the successful transgenic use of R genes critically depends on achieving an optimal level of their expression, possibly in a tissue-specific way. PMID:21438988
Brunner, Susanne; Hurni, Severine; Herren, Gerhard; Kalinina, Olena; von Burg, Simone; Zeller, Simon L; Schmid, Bernhard; Winzeler, Michael; Keller, Beat
Establishment and characterization of a new mammary adenocarcinoma cell line derived from MMTV neu transgenic mice
A new murine cell line, named MG1361, was established from mammary adenocarcinomas arising in a MMTV-neu transgenic mouse lineage where breast tumors develop in 100% of females, due to the over-expression of the activated rat neu oncogene in the mammary gland. The MG1361 cell line shows an epithelial-like morphology, has a poor plating efficiency, low clonogenic capacity, and a doubling
Maria Grazia Sacco; Laura Gribaldo; Ottavia Barbieri; Gino Turchi; Ileana Zucchi; Angelo Collotta; Luca Bagnasco; Domenico Barone; Cristina Montagna; Anna Villa; Erminio Marafante; Paolo Vezzoni
Assessment of fecundity and germ line transmission in two transgenic pig lines produced by sleeping beauty transposition.
Recently, we described a simplified injection method for producing transgenic pigs using a non-autonomous Sleeping Beauty transposon system. The founder animals showed ubiquitous expression of the Venus fluorophore in almost all cell types. To assess, whether expression of the reporter fluorophore affects animal welfare or fecundity, we analyzed reproductive parameters of two founder boars, germ line transmission, and organ and cell specific transgene expression in animals of the F1 and F2 generation. Molecular analysis of ejaculated sperm cells suggested three monomeric integrations of the Venus transposon in both founders. To test germ line transmission of the three monomeric transposon integrations, wild-type sows were artificially inseminated. The offspring were nursed to sexual maturity and hemizygous lines were established. A clear segregation of the monomeric transposons following the Mendelian rules was observed in the F1 and F2 offspring. Apparently, almost all somatic cells, as well as oocytes and spermatozoa, expressed the Venus fluorophore at cell-type specific levels. No detrimental effects of Venus expression on animal health or fecundity were found. Importantly, all hemizygous lines expressed the fluorophore in comparable levels, and no case of transgene silencing or variegated expression was found after germ line transmission, suggesting that the insertions occurred at transcriptionally permissive loci. The results show that Sleeping Beauty transposase-catalyzed transposition is a promising approach for stable genetic modification of the pig genome. PMID:24705079
Garrels, Wiebke; Holler, Stephanie; Cleve, Nicole; Niemann, Heiner; Ivics, Zoltan; Kues, Wilfried A
A transgenic insect cell line engineered to produce CMP-sialic acid and sialylated glycoproteins
We have previously engineered transgenic insect cell lines to express mammalian glycosyltransferases and showed that these cells can sialylate N-glycoproteins, despite the fact that they have little intracellular sialic acid and no detectable CMP¢sialic acid. In the accompanying study, we presented evidence that these cell lines can salvage sialic acids for de novo glycoprotein sialylation from extracellular sialogly-coproteins, such as fetuin, found in fetal bovine serum. This finding led us to create a new transgenic insect cell line designed to synthesize its own sialic acid and CMP¢sialic acid. SfSWT-1 cells, which encode five mammalian glycosyltransferases, were transformed with two additional mammalian genes that encode sialic acid synthase and CMP¢sialic acid synthetase. The resulting cell line expressed all seven mammalian genes, produced CMP¢sialic acid, and sialylated a recombinant glycoprotein when cultured in a serum-free growth medium supplemented with N-acetylmannosamine. Thus the addition of mammalian genes encoding two enzymes involved in CMP¢sialic acid biosynthesis yielded a new transgenic insect cell line, SfSWT-3, that can sialylate recombinant glycoproteins in the absence of fetal bovine serum. This new cell line will be widely useful as an improved host for baculovirus-mediated recombinant glycoprotein production.
Aumiller, Jared J.; Hollister, Jason R.; Jarvis, Donald L.
Generation of a germ cell-specific mouse transgenic Cre line, Vasa-Cre
Summary Cell type-specific genetic modification using the Cre/loxP system is a powerful tool for genetic analysis of distinct cell lineages. Because of the exquisite specificity of Vasa expression (confined to the germ cell lineage in invertebrate and vertebrate species), we hypothesized that a Vasa promoter-driven transgenic Cre line would prove useful for the germ cell lineage-specific inactivation of genes. Here we describe a transgenic mouse line, Vasa-Cre, where Cre is efficiently and specifically expressed in germ cells. Northern analysis showed that transgene expression was confined to the gonads. Cre-mediated recombination with the Rosa26-lacZ reporter was observed beginning at ~e15, and was >95% efficient in male and female germ cells by birth. There was no ectopic activity in most adults, although some animals showed more widespread lacZ expression. This Vasa-Cre transgenic line should thus prove useful for genetic analysis of diverse aspects of germ cell function and gametogenesis.
Gallardo, Teresa; Shirley, Lane; John, George; Castrillon, Diego H.
Production of fat-1 transgenic rats using a post-natal female germline stem cell line.
Germline stem cell lines possess the abilities of self-renewal and differentiation, and have been established from both mouse and human ovaries. Here, we established a new female germline stem cell (FGSC) line from post-natal rats by immunomagnetic sorting for Fragilis, which showed a normal karyotype, high telomerase activity, and a consistent gene expression pattern of primordial germ cells after 1 year of culture. Using an in vitro differentiation system, the FGSC line could differentiate into oocytes. After liposome-based transfection with green fluorescent protein (GFP) or fat-1 vectors, the FGSCs were transplanted into the ovaries of infertile rats. The transplanted FGSCs underwent oogenesis, and the rats produced offspring carrying the GFP or fat-1 transgene after mating with wild-type male rats. The efficiency of gene transfer was 27.86-28.00%, and 2 months was needed to produce transgenic rats. These findings have implications in biomedical research and potential applications in biotechnology. PMID:24258451
Zhou, Li; Wang, Lei; Kang, Jing X; Xie, Wenhai; Li, Xiaoyong; Wu, Changqing; Xu, Bo; Wu, Ji
Transgene flow to hybrid rice and its male-sterile lines.
Gene flow from genetically modified (GM) crops to the same species or wild relatives is a major concern in risk assessment. Transgenic rice with insect and/or disease resistance, herbicide, salt and/or drought tolerance and improved quality has been successfully developed. However, data on rice gene flow from environmental risk assessment studies are currently insufficient for the large-scale commercialization of GM rice. We have provided data on the gene flow frequency at 17 distances between a GM japonica line containing the bar gene as a pollen donor and two indica hybrid rice varieties and four male-sterile (ms) lines. The GM line was planted in a 640 m2 in an isolated experimental plot (2.4 ha), which simulates actual conditions of rice production with pollen competition. Results showed that: (1) under parallel plantation at the 0-m zone, the transgene flow frequency to the ms lines ranged from 3.145 to 36.116% and was significantly higher than that to hybrid rice cultivars (0.037-0.045%). (2) Gene flow frequency decreased as the distance increased, with a sharp cutoff point at about 1-2 m; (3) The maximum distance of transgene flow was 30-40 m to rice cultivars and 40-150 m to ms lines. We believe that these data will be useful for the risk assessment and management of transgenic rice lines, especially in Asia where 90% of world's rice is produced and hybrid rice varieties are extensively used. PMID:17443417
Jia, Shirong; Wang, Feng; Shi, Lei; Yuan, Qianhua; Liu, Wuge; Liao, Yilong; Li, Shuguang; Jin, Wujun; Peng, Huipu
Detection of transgenes in three genetically modified rice lines by fluorescence in situ hybridization
Fluorescence in situ hybridization (FISH) using T-DNA probes was applied to localize transgenes onto specific chromosomes\\u000a and confirm the steady integration of transferred genes in three genetically modified (GM) rice lines, LS28 (event LS30-32-20-1),\\u000a Cry1Ac1 (event C7-1-9-1) and LS28ūCry1Ac1 (event L\\/C1-1-3-1), which are a rice leaf blast-resistant single trait GM line,\\u000a a leaf folder-resistant single trait GM line, and a
Hye Mi Park; Eun Jin Jeon; Nomar Espinosa Waminal; Kong Sik Shin; Soon Jong Kweon; Beom-Seok Park; Seok Cheol Suh; Hyun Hee Kim
Generation and characterization of a GFP transgenic rat line for embryological research
Model organisms expressing fluorescent proteins are important tools for research. The present study was performed to generate\\u000a and characterize a new line of green fluorescent protein (GFP) transgenic rats for use as a model in experimental embryological\\u000a research. We injected a GFP expression vector into 135 zygotes of the Sprague-Dawley (SD) rat strain. Embryo transfer of 103\\u000a surviving embryos resulted
Elena Popova; Brit Rentzsch; Michael Bader; Alexander Krivokharchenko
Prenatal Lethality in a Transgenic Mouse Line is the Result of a Chromosomal Translocation
We have produced a line of transgenic mice that is characterized by prenatal lethality. These mice bear a chimeric plasmid containing the long terminal repeat of the Rous sarcoma virus linked to the coding region of the chloramphenicol acetyltransferase gene (pRSV-CAT). Mice heterozygous for the pRSV-CAT integration site are semisterile, producing litters ≈ 40% of the average size when crossed
Kathleen A. Mahon; Paul A. Overbeek; Heiner Westphal
Retinal Degeneration in Two Lines of Transgenic S334ter Rats
Aim of this study was to examine synaptic connectivity changes in the retina and the location and rate of apoptosis in transgenic S334ter line-3 and line-5 rats with photoreceptor degeneration. Heterozygous S334ter-line-3 and line-5 at P11-13, P30, P60, P90 and several control non-dystrophic rats (Long Evans and Sprague-Dawley) at P60, were studied anatomically by immunohistochemistry for various cell and synaptic markers, and by PNA and TUNEL label.- S334ter line-3 exhibited the fastest rate of degeneration with an early loss of photoreceptors, with 1¢2 layers remaining at P30, and only cones left at P60. Line-5 had 4¢5 layers left at P30, and very few rods left at P60-90. In both lines, horizontal cell processes (including dendrites and axon) were diminished at P11-13, showing gaps in the outer plexiform layer (OPL) at P60, and at P90, almost no terminal tips could be seen. Bipolar cells showed a retraction of their dendrites forming clusters along the OPL. Synaptic terminals of A-II amacrine cells in the IPL lost most of their parvalbumin-immunoreactivity. The apoptosis rate was different in both lines. Line-3 rats showed many photoreceptors affected at P11, occupying the innermost part of the outer nuclear layer. Line-5 showed a lower number of apoptotic cells within the same location at P13. In summary, the S334ter line-3 rat has a faster progression of degeneration than line-5. The horizontal and bipolar terminals are already affected at P11-P13 in both models. Apoptosis is related to the mutated rhodopsin transgene; the first photoreceptor cells affected are those more exposed to light close to the OPL.
Martinez-Navarrete, G.; Seiler, M.J.; Aramant, R.B.; Fernandez-Sanchez, L.; Pinilla, I.; Cuenca, N.
Germ-Line Recombination Activity of the Widely Used hGFAP-Cre and Nestin-Cre Transgenes
Herein we demonstrate with PCR, immunodetection and reporter gene approaches that the widely used human Glial Fibrillary Acidic Protein (hGFAP)-Cre transgene exhibits spontaneous germ-line recombination activity in leading to deletion in brain, heart and tail tissue with high frequency. The ectopic activity of hGFAP-Cre requires a rigorous control. We likewise observed that a second widely used nestin-Cre transgene shows germ-line deletion. Here we describe procedures to identify mice with germ-line recombination mediated by the hGFAP-Cre and nestin-Cre transgenes. Such control is essential to avoid pleiotropic effects due to germ-line deletion of loxP-flanked target genes and to maintain the CNS-restricted deletion status in transgenic mouse colonies.
Zhang, Jiong; Dublin, Pavel; Griemsmann, Stephanie; Klein, Alexandra; Brehm, Ralph; Bedner, Peter; Fleischmann, Bernd K.; Steinhauser, Christian; Theis, Martin
A Phox2b::FLPo transgenic mouse line suitable for intersectional genetics
Phox2b is a transcription factor expressed in the central and peripheral neurons that control cardiovascular, respiratory and digestive functions and essential for their development. Several populations known or suspected to regulate visceral functions express Phox2b in the developing hindbrain. Extensive cell migration and lack of suitable markers have greatly hampered studying their development. Reasoning that intersectional fate mapping may help to overcome these impediments, we have generated a BAC transgenic mouse line, P2b::FLPo, which expresses codon-optimized FLP recombinase in Phox2b expressing cells. By partnering the P2b::FLPo with the FLP-responsive RC::Fela allele, we show that FLP recombination switches on lineage tracers in the cells that express or have expressed Phox2b, permanently marking them for study across development. Taking advantage of the dualrecombinase feature of RC::Fela, we further show that the P2b::FLPo transgene can be partnered with Lbx1Cre as Cre driver to generate triple transgenics in which neurons having a history of both Phox2b and Lbx1 expression are specifically labelled. Hence, the P2b::FLPo line when partnered with a suitable Cre driver provides a tool for tracking and accessing genetically subsets of Phox2b-expressing neuronal populations, which has not been possible by Cremediated recombination alone.
Hirsch, Marie-Rose; d'Autreaux, Fabien; Dymecki, Susan M.; Brunet, Jean-Francois; Goridis, Christo
Transgenic mouse lines expressing synaptopHluorin in hippocampus and cerebellar cortex.
We generated six transgenic mouse lines in which synaptopHluorin (SpH), one of green fluorescent protein-based sensors of vesicular exocytosis, was expressed under the control of neuron-specific Thy-1.2 promoter. In situ hybridization study revealed that SpH mRNA was expressed in a broad spectrum of brain regions in four of them, whereas in others it was expressed in the specific regions of the hippocampus. In one particular line, SpH immunoreactivity was specifically observed in the mossy fiber presynaptic terminals of both hippocampus and cerebellar cortex. The fluorescence intensity of these presynaptic terminals was somewhat decreased by acidic buffer superfusion and greatly increased by vesicular neutralization of pH, indicating that the SpH molecules are mainly distributed in the synaptic vesicles. The exocytosis-dependent fluorescence increment was measured upon activation of a single presynaptic terminal. These transgenic lines are expected to facilitate morphological and physiological studies of presynaptic terminals in a variety of regions of the brain. PMID:15880564
Araki, Rikita; Sakagami, Hiroyuki; Yanagawa, Yuchio; Hikima, Takuya; Ishizuka, Toru; Yawo, Hiromu
Transgenic mouse lines for non-invasive ratiometric monitoring of intracellular chloride.
Chloride is the most abundant physiological anion and participates in a variety of cellular processes including trans-epithelial transport, cell volume regulation, and regulation of electrical excitability. The development of tools to monitor intracellular chloride concentration ([Cli]) is therefore important for the evaluation of cellular function in normal and pathological conditions. Recently, several Cl-sensitive genetically encoded probes have been described which allow for non-invasive monitoring of [Cli]. Here we describe two mouse lines expressing a CFP-YFP-based Cl probe called Cl-Sensor. First, we generated transgenic mice expressing Cl-Sensor under the control of the mouse Thy1 mini promoter. Cl-Sensor exhibited good expression from postnatal day two (P2) in neurons of the hippocampus and cortex, and its level increased strongly during development. Using simultaneous whole-cell monitoring of ionic currents and Cl-dependent fluorescence, we determined that the apparent EC 50 for Cli was 46 mM, indicating that this line is appropriate for measuring neuronal [Cli] in postnatal mice. We also describe a transgenic mouse reporter line for Cre-dependent conditional expression of Cl-Sensor, which was targeted to the Rosa26 locus and by incorporating a strong exogenous promoter induced robust expression upon Cre-mediated recombination. We demonstrate high levels of tissue-specific expression in two different Cre-driver lines targeting cells of the myeloid lineage and peripheral sensory neurons. Using these mice the apparent EC 50 for Cli was estimated to be 61 and 54 mM in macrophages and DRG, respectively. Our data suggest that these mouse lines will be useful models for ratiometric monitoring of Cli in specific cell types in vivo. PMID:23734096
Batti, Laura; Mukhtarov, Marat; Audero, Enrica; Ivanov, Anton; Paolicelli, Rosa Chiara; Zurborg, Sandra; Gross, Cornelius; Bregestovski, Piotr; Heppenstall, Paul A
Transgenic mouse lines for non-invasive ratiometric monitoring of intracellular chloride
Chloride is the most abundant physiological anion and participates in a variety of cellular processes including trans-epithelial transport, cell volume regulation, and regulation of electrical excitability. The development of tools to monitor intracellular chloride concentration ([Cli]) is therefore important for the evaluation of cellular function in normal and pathological conditions. Recently, several Cl-sensitive genetically encoded probes have been described which allow for non-invasive monitoring of [Cli]. Here we describe two mouse lines expressing a CFP-YFP-based Cl probe called Cl-Sensor. First, we generated transgenic mice expressing Cl-Sensor under the control of the mouse Thy1 mini promoter. Cl-Sensor exhibited good expression from postnatal day two (P2) in neurons of the hippocampus and cortex, and its level increased strongly during development. Using simultaneous whole-cell monitoring of ionic currents and Cl-dependent fluorescence, we determined that the apparent EC50 for Cli was 46 mM, indicating that this line is appropriate for measuring neuronal [Cli] in postnatal mice. We also describe a transgenic mouse reporter line for Cre-dependent conditional expression of Cl-Sensor, which was targeted to the Rosa26 locus and by incorporating a strong exogenous promoter induced robust expression upon Cre-mediated recombination. We demonstrate high levels of tissue-specific expression in two different Cre-driver lines targeting cells of the myeloid lineage and peripheral sensory neurons. Using these mice the apparent EC50 for Cli was estimated to be 61 and 54 mM in macrophages and DRG, respectively. Our data suggest that these mouse lines will be useful models for ratiometric monitoring of Cli in specific cell types in vivo.
Batti, Laura; Mukhtarov, Marat; Audero, Enrica; Ivanov, Anton; Paolicelli, Rosa Chiara; Zurborg, Sandra; Gross, Cornelius; Bregestovski, Piotr; Heppenstall, Paul A.
Characterization of Prostatic Epithelial Cell Lines Derived from Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP) Model1
To develop a syngeneic transplantablesystem to study immunothera peutic approaches for the treatment of prostate cancer, three cell lines were established from a heterogeneous 32 week tumor of the t@ansgenic adenocarcinoma g@ouseprostate (TRAMP) modeL TRAMP is a transgenic line of C57B116 mice harboring a construct comprised of the minimal ŌĆ\\
Barbara A. Foster; Jeffrey R. Gingrich; Eugene D. Kwon; Christopher Madias; Norman M. Greenberg
Transgene expression and silencing in a tick cell line: A model system for functional tick genomics.
The genome project of the black legged tick, Ixodes scapularis, provides sequence data for testing gene function and regulation in this important pathogen vector. We tested Sleeping Beauty (SB), a Tc1/mariner group transposable element, and cationic lipid-based transfection reagents for delivery and genomic integration of transgenes into I. scapularis cell line ISE6. Plasmid DNA and dsRNA were effectively transfected into ISE6 cells and they were successfully transformed to express a red fluorescent protein (DsRed2) and a selectable marker, neomycin phosphotransferase (NEO). Frequency of transformation was estimated as 1 transformant per 5000-10,000 cells and cultures were incubated for 2-3 months in medium containing the neomycin analog G418 in order to isolate transformants. Genomic integration of the DsRed2 transgene was confirmed by inverse PCR and sequencing that demonstrated a TA nucleotide pair inserted between SB inverted/direct repeat sequences and tick genomic sequences, indicating that insertion of the DsRed2 gene into the tick cell genome occurred through the activity of SB transposase. RNAi using dsRNA transcribed from the DsRed2 gene silenced expression of red fluorescent protein in transformed ISE6 cells. SB transposition in cell line ISE6 provides an effective means to explore the functional genomics of I. scapularis. PMID:18722527
Kurtti, Timothy J; Mattila, Joshua T; Herron, Michael J; Felsheim, Roderick F; Baldridge, Gerald D; Burkhardt, Nicole Y; Blazar, Bruce R; Hackett, Perry B; Meyer, Jason M; Munderloh, Ulrike G
Germ-line transmission of lentiviral PGK-EGFP integrants in transgenic cattle: new perspectives for experimental embryology.
Lentiviral vectors are a powerful tool for the genetic modification of livestock species. We previously generated transgenic founder cattle with lentiviral integrants carrying enhanced green fluorescent protein (EGFP) under the control of the phosphoglycerate kinase (PGK) promoter. In this study, we investigated the transmission of LV-PGK-EGFP integrants through the female and male germ line in cattle. A transgenic founder heifer (#562, Kiki) was subjected to superovulation treatment and inseminated with semen from a non-transgenic bull. Embryos were recovered and transferred to synchronized recipient heifers, resulting in the birth of a healthy male transgenic calf expressing EGFP as detected by in vivo imaging. Semen from a transgenic founder bull (#561, Jojo) was used for in vitro fertilization (IVF) of in vitro matured (IVM) oocytes from non-transgenic cows. The rates of cleavage and development to blastocyst in vitro corresponded to 52.0 +/- 4.1 and 24.5 +/- 4.4%, respectively. Expression of EGFP was observed at blastocyst stage (day 7 after IVF) and was seen in 93.0% (281/302) of the embryos. 24 EGFP-expressing embryos were transferred to 9 synchronized recipients. Analysis of 2 embryos, flushed from the uterus on day 15, two fetuses recovered on day 45, and a healthy male transgenic calf revealed consistent high-level expression of EGFP in all tissues investigated. Our study shows for the first time transmission of lentiviral integrants through the germ line of female and male transgenic founder cattle. The pattern of inheritance was consistent with Mendelian rules. Importantly, high fidelity expression of EGFP in embryos, fetuses, and offspring of founder #561 provides interesting tools for developmental studies in cattle, including interactions of gametes, embryos and fetuses with their maternal environment. PMID:19862638
Reichenbach, Myriam; Lim, Tiongti; Reichenbach, Horst-Dieter; Guengoer, Tuna; Habermann, Felix A; Matthiesen, Marieke; Hofmann, Andreas; Weber, Frank; Zerbe, Holm; Grupp, Thomas; Sinowatz, Fred; Pfeifer, Alexander; Wolf, Eckhard
Beneficial 'unintended effects' of a cereal cystatin in transgenic lines of potato, Solanum tuberosum
Background Studies reported unintended pleiotropic effects for a number of pesticidal proteins ectopically expressed in transgenic crops, but the nature and significance of such effects in planta remain poorly understood. Here we assessed the effects of corn cystatin II (CCII), a potent inhibitor of C1A cysteine (Cys) proteases considered for insect and pathogen control, on the leaf proteome and pathogen resistance status of potato lines constitutively expressing this protein. Results The leaf proteome of lines accumulating CCII at different levels was resolved by 2-dimensional gel electrophoresis and compared with the leaf proteome of a control (parental) line. Out of ca. 700 proteins monitored on 2-D gels, 23 were significantly up- or downregulated in CCII-expressing leaves, including 14 proteins detected de novo or up-regulated by more than five-fold compared to the control. Most up-regulated proteins were abiotic or biotic stress-responsive proteins, including different secretory peroxidases, wound inducible protease inhibitors and pathogenesis-related proteins. Accordingly, infection of leaf tissues by the fungal necrotroph Botryris cinerea was prevented in CCII-expressing plants, despite a null impact of CCII on growth of this pathogen and the absence of extracellular Cys protease targets for the inhibitor. Conclusions These data point to the onset of pleiotropic effects altering the leaf proteome in transgenic plants expressing recombinant protease inhibitors. They also show the potential of these proteins as ectopic modulators of stress responses in planta, useful to engineer biotic or abiotic stress tolerance in crop plants of economic significance.
Phototropism and gravitropism in transgenic lines of Arabidopsis altered in the phytochrome pathway.
Phytochromes are a family of photoreceptor molecules, absorbing primarily in red and far-red, that are important in many aspects of plant development. These studies investigated the role of phytochromes in phototropism and gravitropism of seedlings of Arabidopsis thaliana. We used two transgenic lines, one which lacked phytochromes specifically in the roots (M0062/UASBVR) and the other lacked phytochromes in the shoots (CAB3::pBVR). These transgenic plants are deficient in the phytochrome chromophore in specific tissues due the expression of biliverdin IXa reductase (BVR), which binds to precursors of the chromophore. Experiments were performed in both light and dark conditions to determine whether roots directly perceive light signals or if the signal is perceived in the shoot and then transmitted to the root during tropistic curvature. Kinetics of tropisms and growth were assayed by standard methods or with a computer-based feedback system. We found that the perception of red light occurs directly in the root during phototropism in this organ and that signaling also may occur from root to shoot in gravitropism. PMID:22380624
Hopkins, Jane A; Kiss, John Z
De novo piRNA cluster formation in the Drosophila germ line triggered by transgenes containing a transcribed transposon fragment
PIWI-interacting RNAs (piRNAs) provide defence against transposable element (TE) expansion in the germ line of metazoans. piRNAs are processed from the transcripts encoded by specialized heterochromatic clusters enriched in damaged copies of transposons. How these regions are recognized as a source of piRNAs is still elusive. The aim of this study is to determine how transgenes that contain a fragment of the Long Interspersed Nuclear Elements (LINE)-like I transposon lead to an acquired TE resistance in Drosophila. We show that such transgenes, being inserted in unique euchromatic regions that normally do not produce small RNAs, become de novo bidirectional piRNA clusters that silence I-element activity in the germ line. Strikingly, small RNAs of both polarities are generated from the entire transgene and flanking genomic sequencesŚnot only from the transposon fragment. Chromatin immunoprecipitation analysis shows that in ovaries, the trimethylated histone 3 lysine 9 (H3K9me3) mark associates with transgenes producing piRNAs. We show that transgene-derived hsp70 piRNAs stimulate in trans cleavage of cognate endogenous transcripts with subsequent processing of the non-homologous parts of these transcripts into piRNAs.
Olovnikov, Ivan; Ryazansky, Sergei; Shpiz, Sergey; Lavrov, Sergey; Abramov, Yuri; Vaury, Chantal; Jensen, Silke; Kalmykova, Alla
zebraflash transgenic lines for in vivo bioluminescence imaging of stem cells and regeneration in adult zebrafish
The zebrafish has become a standard model system for stem cell and tissue regeneration research, based on powerful genetics, high tissue regenerative capacity and low maintenance costs. Yet, these studies can be challenged by current limitations of tissue visualization techniques in adult animals. Here we describe new imaging methodology and present several ubiquitous and tissue-specific luciferase-based transgenic lines, which we have termed zebraflash, that facilitate the assessment of regeneration and engraftment in freely moving adult zebrafish. We show that luciferase-based live imaging reliably estimates muscle quantity in an internal organ, the heart, and can longitudinally follow cardiac regeneration in individual animals after major injury. Furthermore, luciferase-based detection enables visualization and quantification of engraftment in live recipients of transplanted hematopoietic stem cell progeny, with advantages in sensitivity and gross spatial resolution over fluorescence detection. Our findings present a versatile resource for monitoring and dissecting vertebrate stem cell and regeneration biology.
Chen, Chen-Hui; Durand, Ellen; Wang, Jinhu; Zon, Leonard I.; Poss, Kenneth D.
Rapid construction of transgene-amplified CHO cell lines by cell cycle checkpoint engineering.
The process of establishing high-producing cell lines for the manufacture of therapeutic proteins is usually both time-consuming and laborious due to the low probability of obtaining high-producing clones from a pool of transfected cells and slow cell growth under the strong selective pressure of screening to identify high-producing clones. We present a novel method to rapidly generate more high-producing cells by accelerating transgene amplification. A small interfering RNA (siRNA) expression vector against ataxia telangiectasia and Rad3 related (ATR), a cell cycle checkpoint kinase, was transfected into Chinese hamster ovary (CHO) cells. The influences of ATR downregulation on gene amplification and the productivity were investigated in CHO cells producing green fluorescent protein (GFP) and secreting monoclonal antibody (mAb). The ATR-downregulated cells showed up to a 6-fold higher ratio of GFP-positive cells than that of the control cell pool. Moreover, the downregulated mAb-producing cells had about a 4-fold higher specific production rate and a 3-fold higher volumetric productivity as compared with the mock cells. ATR-downregulated cells showed a much faster increase in transgene copy numbers during the gene amplification process via methotrexate (MTX) treatment in both GFP- and mAb-producing cells. Our results suggest that a pool of high-producing cells can be more rapidly generated by ATR downregulation as compared with conventional gene amplification by MTX treatment. This novel method may be a promising approach to reduce time and labor in the process of cell line development. PMID:23615744
Lee, Kyoung Ho; Onitsuka, Masayoshi; Honda, Kohsuke; Ohtake, Hisao; Omasa, Takeshi
Analysis of T-DNA/Host-Plant DNA Junction Sequences in Single-Copy Transgenic Barley Lines
Sequencing across the junction between an integrated transfer DNA (T-DNA) and a host plant genome provides two important pieces of information. The junctions themselves provide information regarding the proportion of T-DNA which has integrated into the host plant genome, whilst the transgene flanking sequences can be used to study the local genetic environment of the integrated transgene. In addition, this information is important in the safety assessment of GM crops and essential for GM traceability. In this study, a detailed analysis was carried out on the right-border T-DNA junction sequences of single-copy independent transgenic barley lines. T-DNA truncations at the right-border were found to be relatively common and affected 33.3% of the lines. In addition, 14.3% of lines had rearranged construct sequence after the right border break-point. An in depth analysis of the host-plant flanking sequences revealed that a significant proportion of the T-DNAs integrated into or close to known repetitive elements. However, this integration into repetitive DNA did not have a negative effect on transgene expression.
Bartlett, Joanne G.; Smedley, Mark A.; Harwood, Wendy A.
An ?-smooth muscle actin (acta2/?sma) zebrafish transgenic line marking vascular mural cells and visceral smooth muscle cells.
Mural cells of the vascular system include vascular smooth muscle cells (SMCs) and pericytes whose role is to stabilize and/or provide contractility to blood vessels. One of the earliest markers of mural cell development in vertebrates is ? smooth muscle actin (acta2; ?sma), which is expressed by pericytes and SMCs. In vivo models of vascular mural cell development in zebrafish are currently lacking, therefore we developed two transgenic zebrafish lines driving expression of GFP or mCherry in acta2-expressing cells. These transgenic fish were used to trace the live development of mural cells in embryonic and larval transgenic zebrafish. acta2:EGFP transgenic animals show expression that largely mirrors native acta2 expression, with early pan-muscle expression starting at 24 hpf in the heart muscle, followed by skeletal and visceral muscle. At 3.5 dpf, expression in the bulbus arteriosus and ventral aorta marks the first expression in vascular smooth muscle. Over the next 10 days of development, the number of acta2:EGFP positive cells and the number of types of blood vessels associated with mural cells increases. Interestingly, the mural cells are not motile and remain in the same position once they express the acta2:EGFP transgene. Taken together, our data suggests that zebrafish mural cells develop relatively late, and have little mobility once they associate with vessels. PMID:24594685
Whitesell, Thomas R; Kennedy, Regan M; Carter, Alyson D; Rollins, Evvi-Lynn; Georgijevic, Sonja; Santoro, Massimo M; Childs, Sarah J
Inositol based non-viral vectors for transgene expression in human cervical carcinoma and hepatoma cell lines.
Myo-Inositol (INO) is a biomolecule with crucial functions in many aspects. In this study, hyperbranched copolymers for gene delivery were synthesized based on inositol and low molecular weight polyethylenimine. The capacity of INO-PEIs to load plasmid DNA and their biocompatibility was demonstrated. A tumor target ligand, folic acid (FA), which was widely used for drug delivery systems, was subsequently conjugated to INO-PEIs and resulted in INO-PEI-FA copolymers. The polymers were then evaluated on their activity to mediate transgene expression in mammalian cell lines. As indicated, INO-PEIs were able to mediate efficient transgene expression, which was particularly noticeable in carcinoma cell line HeLa. INO-PEI-FA further improved the efficiency in HepG2. Distribution of INO-PEI-FA polymers in non-carcinoma NIH 3T3 and carcinoma HeLa cell lines was discussed. PMID:24314555
Zhang, Lei; Fan, Yiwen; Wu, Yunkun
Establishment and characterization of differentiated, nontransformed hepatocyte cell lines derived from mice transgenic for transforming growth factor alpha.
Hepatocytes are extensively used in studies of gene regulation but cannot be maintained in long-term culture as replicating, differentiated cells while remaining nontumorigenic. We have derived two hepatocyte lines from livers of transgenic mice overexpressing transforming growth factor alpha, a potent hepatocyte mitogen, which overcome these limitations. The transgenic hepatocytes were maintained for > or = 2 months in serum-supplemented primary culture and gave rise to cell lines, of which two (AML12 and AML14) have been cultured for > 1.5 years (> 80 passages). Both lines have typical hepatocyte features such as peroxisomes and bile canalicular-like structures, do not grow in soft agar, and are nontumorigenic in nude mice. Like normal hepatocytes, AML cells express high levels of mRNA for serum (albumin, alpha 1-antitrypsin, and transferrin) and gap junction (connexins 26 and 32) proteins, secrete albumin, and contain solely isozyme 5 of lactate dehydrogenase. After extensive passaging, AML12 cells continue to strongly coexpress hepatocyte connexin mRNAs but do not display nonparenchymal cell markers. Although mRNA levels for some serum proteins progressively fall, high expression in late AML12 cultures may be regained by passage in serum-free medium. The AML14 line loses expression of both differentiated markers and transgene mRNA with extended passaging, and hepatocytic traits are only partially restored by passage in serum-free medium. These differentiated, nontumorigenic cell lines should serve as models in which to study hepatocyte growth and differentiation. PMID:7904757
Wu, J C; Merlino, G; Fausto, N
Zebrafish Transgenic Line huORFZ Is an Effective Living Bioindicator for Detecting Environmental Toxicants
Reliable animal models are invaluable for monitoring the extent of pollution in the aquatic environment. In this study, we demonstrated the potential of huORFZ, a novel transgenic zebrafish line that harbors a human upstream open reading frame of the chop gene fused with GFP reporter, as an animal model for monitoring environmental pollutants and stress-related cellular processes. When huORFZ embryos were kept under normal condition, no leaked GFP signal could be detected. When treated with hazardous chemicals, including heavy metals and endocrine-disrupting chemicals near their sublethal concentrations (LC50), huORFZ embryos exhibited different tissue-specific GFP expression patterns. For further analysis, copper (Cu2+), cadmium (Cd2+) and Chlorpyrifos were applied. Cu2+ triggered GFP responses in skin and muscle, whereas Cd2+ treatment triggered GFP responses in skin, olfactory epithelium and pronephric ducts. Moreover, fluorescence intensity, as exhibited by huORFZ embryos, was dose-dependent. After surviving treated embryos were returned to normal condition, survival rates, as well as TUNEL signals, returned to pretreatment levels with no significant morphological defects observed. Such results indicated the reversibility of treatment conditions used in this study, as long as embryos survived such conditions. Notably, GFP signals decreased along with recovery, suggesting that GFP signaling of huORFZ embryos likely reflected the overall physiological condition of the individual. To examine the performance of the huORFZ line under real-world conditions, we placed huORFZ embryos in different river water samples. We found that the huORFZ embryos correctly detected the presence of various kinds of pollutants. Based on these findings, we concluded that such uORFchop-based system can be integrated into a first-line water alarm system monitoring the discharge of hazardous pollutants.
Chu, Chien; Li, Hong-Ping; Tsai, Huai-Jen
Adeno-associated virus (AAV)-mediated transduction of male germ line stem cells results in transgene transmission after germ cell transplantation
We explored whether exposure of mam- malian germ line stem cells to adeno-associated virus (AAV), a gene therapy vector, would lead to stable transduction and transgene transmission. Mouse germ cells harvested from experimentally induced crypt- orchid donor testes were exposed in vitro to AAV vectors carrying a GFP transgene and transplanted to germ cell-depleted syngeneic recipient testes, resulting in colonization
Ali Honaramooz; Susan Megee; Wenxian Zeng; Margret M. Destrempes; Susan A. Overton; Jinping Luo; Hannah Galantino-Homer; Mark Modelski; Fangping Chen; Stephen Blash; David T. Melican; William G. Gavin; Sandra Ayres; Fang Yang; P. Jeremy Wang; Yann Echelard; Ina Dobrinski
Strain and gender differences in the behavior of mouse lines commonly used in transgenic studies.
The present study was aimed at establishing behavioral differences between three inbred mouse strains (129S2/SvHsd, C57BL/6JOlaHsd, FVB/NHsd) and two F1 hybrid lines derived from them (129 x C57BL/6 and 129 x FVB). The choice of the given strains was based on the frequent use of these mice in transgenic research. For the behavioral phenotyping, we employed a test battery consisting of the following models: elevated plus-maze (EPM), open field (OF), light-dark exploration, spontaneous locomotor activity, rota-rod (RR), Porsolt's forced-swimming test (FST), and Morris water task. Significant variations between the strains were established in all tests. Anxiety-like behavior was more pronounced in the 129S2/Sv and 129 x C57BL/6 mice, the FVB/N mice were spontaneously hyperactive, the best coordination ability was demonstrated by the C57BL/6 and 129 x C57BL/6 groups. A good performance in the learning test was established in both hybrid lines and the 129S2/Sv mice, whereas the well-known visual impairment of the FVB strain was confirmed by low performance in spatial and non-spatial tasks. Differences related to the gender were revealed occasionally; most importantly, 129 x C57BL/6 males had a higher anxiety level than their female counterparts in the EPM. Several other gender dissociations suggest the strain and task specificity. In conclusion, we would like to highlight the importance of the genetic background and gender of mice for the molecular biological and pharmacological studies and also the need for well-established testing protocols to obtain wide information at the first stage of behavioral screening of genetically modified mice. PMID:11240006
V§ikar, V; K§ks, S; Vasar, E; Rauvala, H
Characterization of the ovalbumin-specific TCR transgenic line OT-I: MHC elements for positive and negative selection
The present report provides the first extensive characterization of the OT-I TCR transgenic line, which produces MHC class I-restricted, ovalbumin-specific, CD8+ T cells (OT-I cells). These cells are shown to be positively selected in vivo in H-2b C57BL\\/6 mice and in bm5 mice, which express the Kbm5 mutant molecule. In contrast, OT-I cells were not selected by mutant Kb molecules
Sally RMcK Clarke; Megan Barnden; Christian Kurts; Francis R Carbone; Jacques Fap Miller; William R Heath
c-RET Molecule in Malignant Melanoma from Oncogenic RET-Carrying Transgenic Mice and Human Cell Lines
Malignant melanoma is one of the most aggressive cancers and its incidence worldwide has been increasing at a greater rate than that of any other cancer. We previously reported that constitutively activated RFP-RET-carrying transgenic mice (RET-mice) spontaneously develop malignant melanoma. In this study, we showed that expression levels of intrinsic c-Ret, glial cell line-derived neurotrophic factor (Gdnf) and Gdnf receptor
Yuichiro Ohshima; Ichiro Yajima; Kozue Takeda; Machiko Iida; Mayuko Kumasaka; Yoshinari Matsumoto; Masashi Kato; Benjamin Edward Rich
Establishment of a preadipocyte cell line derived from mature adipocytes of GFP transgenic mice and formation of adipose tissue
We established a preadipocyte cell line from mature adipocytes obtained from subcutaneous fat tissue of green fluorescent\\u000a protein (GFP) transgenic mice. The floating top layer, containing mature adipocytes, was isolated from subcutaneous fat tissue\\u000a by collagenase digestion and filtration. Fluorescence-activated cell sorting and microscopic analysis revealed that the floating\\u000a cell fraction comprised a highly homogeneous adipocyte population with no adipose
Hiroyuki Nobusue; Tsuyoshi Endo; Koichiro Kano
Fast-tracking development of homozygous transgenic cereal lines using a simple and highly flexible real-time PCR assay
Background A crucial step in the evaluation of newly produced transgenic plants is the selection of homozygous plants. Here we describe an efficient and highly flexible real-time PCR-based method for the development of homozygous lines in plant models with complex (multiple) genomes and/or relatively long generation times (>3 months) using direct copy number determinations. Results An existing DNA extraction method was converted into a high-throughput plant leaf DNA extraction procedure yielding DNA suitable for real-time PCR analyses. Highly specific and efficient primer pairs were developed for a bread wheat reference gene (Epsilon Cyclase) and for standard sequence elements in the gene cassette routinely used for cereal transformations (an intron bridge and the Nopaline Synthase terminator). The real-time PCR assay reliably distinguished wheat plants with a single copy of the transgene from individuals with multiple copies or those lacking the transgene. To obtain homozygous lines carrying a unique insertion event as efficiently as possible, T0 plants (plants raised from transformed callus) with a single copy of the transgene were selected and their progeny screened for homozygous plants. Finally, the assay was adapted to work on rice. Conclusions The ability to quickly, easily and accurately quantify the construct copy numbers, as provided by the real-time PCR assay, greatly improved the efficiency and reliability of the selection of homozygous transgenic plants in our case study. We were able to select homozygous plants in early generations, avoiding time-consuming methods such as large scale analysis of segregation patterns of descendants and/or Southern blotting. Additionally, the ability to specifically develop homozygous lines carrying a unique insertion event could be important in avoiding gene silencing due to co-suppression, and if needed assist in the selection of lines suitable for future deregulation. The same primer pairs can be used to quantify many different wheat transgenic events because the construct-specific primer pairs are targeted to standard sequence elements of the cereal gene cassettes, making the method widely applicable in wheat GM research. Moreover, because all procedures described here are standardized, the method may easily be adapted to vectors lacking the target regions used here and/or to other plant models.
Transgene stacking and marker elimination in transgenic rice by sequential Agrobacterium-mediated co-transformation with the same selectable marker gene.
Rice chitinase (chi11) and tobacco osmotin (ap24) genes, which cause disruption of fungal cell wall and cell membrane, respectively, were stacked in transgenic rice to develop resistance against the sheath blight disease. The homozygous marker-free transgenic rice line CoT23 which harboured the rice chi11 transgene was sequentially re-transformed with a second transgene ap24 by co-transformation using an Agrobacterium tumefaciens strain harbouring a single-copy cointegrate vector pGV2260::pSSJ1 and a multi-copy binary vector pBin19?nptII-ap24 in the same cell. pGV2260::pSSJ1 T-DNA carried the hygromycin phosphotransferase (hph) and ?-glucuronidase (gus) genes. pBin19?nptII-ap24 T-DNA harboured the tobacco osmotin (ap24) gene. Co-transformation of the gene of interest (ap24) with the selectable marker gene (SMG, hph) occurred in 12 out of 18 T(0) plants (67%). Segregation of hph from ap24 was accomplished in the T(1) generation in one (line 11) of the four analysed co-transformed plants. The presence of ap24 and chi11 transgenes and the absence of the hph gene in the SMG-eliminated T(1) plants of the line 11 were confirmed by DNA blot analyses. The SMG-free transgenic plants of the line 11 harboured a single copy of the ap24 gene. Homozygous, SMG-free T(2) plants of the transgenic line 11 harboured stacked transgenes, chi11 and ap24. Northern blot analysis of the SMG-free plants revealed constitutive expression of chi11 and ap24. The transgenic plants with stacked transgenes displayed high levels of resistance against Rhizoctonia solani. Thus, we demonstrate the development of transgene-stacked and marker-free transgenic rice by sequential Agrobacterium-mediated co-transformation with the same SMG. PMID:21327387
Ramana Rao, Mangu Venkata; Parameswari, Chidambaram; Sripriya, Rajasekaran; Veluthambi, Karuppannan
Direct derivation of conditionally immortal cell lines from an H-2Kb-tsA58 transgenic mouse.
Studies on cell lines have greatly improved our understanding of many important biological questions. Generation of cell lines is facilitated by the introduction of immortalizing oncogenes into cell types of interest. One gene known to immortalize many different cell types in vitro encodes the simian virus 40 (SV40) large tumor (T) antigen (TAg). To circumvent the need for gene insertion in vitro to generate cell lines, we created transgenic mice harboring the SV40 TAg gene. Since previous studies have shown that TAg expression in transgenic mice is associated with tumorigenesis and aberrant development, we utilized a thermolabile TAg [from a SV40 strain, tsA58, temperature sensitive (ts) for transformation] to reduce the levels of functional TAg present in vivo. To direct expression to a broad range of tissues, we used the mouse major histocompatibility complex H-2Kb promoter, which is both widely active and can be further induced by interferons. tsA58 TAg mRNA was expressed in tissues of all animals harboring the hybrid construct. Development of all tissues was macroscopically normal except for thymus, which consistently showed hyperplasia. Fibroblast and cytokeratin+ thymic epithelial cultures from these mice were readily established without undergoing crisis and were conditionally immortal in their growth; the degree of conditionality was correlated with the levels of tsA58 TAg detected. One strain of H-2Kb-tsA58 mice has been bred through several generations to homozygosity and transmits a functional copy of the transgene. PMID:1711218
Jat, P S; Noble, M D; Ataliotis, P; Tanaka, Y; Yannoutsos, N; Larsen, L; Kioussis, D
Modulation of mitochondrial activity by S-nitrosoglutathione reductase in Arabidopsis thaliana transgenic cell lines.
The enzyme S-nitrosoglutathione reductase (GSNOR) has an important role in the metabolism of S-nitrosothiols (SNO) and, consequently, in the modulation of nitric oxide (NO)-mediated processes. Although the mitochondrial electron transport chain is an important target of NO, the role of GSNOR in the functionality of plant mitochondria has not been addressed. Here, we measured SNO content and NO emission in Arabidopsis thaliana cell suspension cultures of wild-type (WT) and GSNOR overexpressing (GSNOR(OE)) or antisense (GSNOR(AS)) transgenic lines, grown under optimal conditions and under nutritional stress. Respiratory activity of isolated mitochondria and expression of genes encoding for mitochondrial proteins were also analyzed. Under optimal growth conditions, GSNOR(OE) had the lowest SNO and NO levels and GSNOR(AS) the highest, as expected by the GSNO-consuming activity of GSNOR. Under stress, this pattern was reversed. Analysis of oxygen uptake by isolated mitochondria showed that complex I and external NADH dehydrogenase activities were inhibited in GSNOR(OE) cells grown under nutritional stress. Moreover, GSNOR(OE) could not increase alternative oxidase (AOX) activity under nutritional stress. GSNOR(AS) showed constitutively high activity of external NADH dehydrogenase, and maintained the activity of the uncoupling protein (UCP) under stress. The alterations observed in mitochondrial protein activities were not strictly correlated to changes in gene expression, but instead seemed to be related with the SNO/NO content, suggesting a post-transcriptional regulation. Overall, our results highlight the importance of GSNOR in modulating SNO and NO homeostasis as well mitochondrial functionality, both under normal and adverse conditions in A. thaliana cells. PMID:23201478
Frungillo, Lucas; de Oliveira, Jusceley Fatima Palamim; Saviani, Elzira Elisabeth; Oliveira, Halley Caixeta; MartĒnez, M Carmen; Salgado, Ione
Visualization of craniofacial development in the sox10: kaede transgenic zebrafish line using time-lapse confocal microscopy.
Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, convergence and extension of facial prominences, and maturation of the craniofacial skeleton. To study the contribution of the cranial neural crest to specific regions of the zebrafish palate a sox10: kaede transgenic zebrafish line was generated. Sox10 provides lineage restriction of the kaede reporter protein to the neural crest, thereby making the cell labeling a more precise process than traditional dye or reporter mRNA injection. Kaede is a photo-convertible protein that turns from green to red after photo activation and makes it possible to follow cells precisely. The sox10: kaede transgenic line was used to perform lineage analysis to delineate CNC cell populations that give rise to maxillary versus mandibular elements and illustrate homology of facial prominences to amniotes. This protocol describes the steps to generate a live time-lapse video of a sox10: kaede zebrafish embryo. Development of the ethmoid plate will serve as a practical example. This protocol can be applied to making a time-lapse confocal recording of any kaede or similar photoconvertible reporter protein in transgenic zebrafish. Furthermore, it can be used to capture not only normal, but also abnormal development of craniofacial structures in the zebrafish mutants. PMID:24121214
Gfrerer, Lisa; Dougherty, Max; Liao, Eric C
Generation of an ABCG2{sup GFPn-puro} transgenic line - A tool to study ABCG2 expression in mice
The ATP-binding cassette (ABC) transporter 2 (ABCG2) is expressed by stem cells in many organs and in stem cells of solid tumors. These cells are isolated based on the side population (SP) phenotype, a Hoechst 3342 dye efflux property believed to be conferred by ABCG2. Because of the limitations of this approach we generated transgenic mice that express Nuclear GFP (GFPn) coupled to the Puromycin-resistance gene, under the control of ABCG2 promoter/enhancer sequences. We show that ABCG2 is expressed in neural progenitors of the developing forebrain and spinal cord and in embryonic and adult endothelial cells of the brain. Using the neurosphere assay, we isolated tripotent ABCG2-expressing neural stem cells from embryonic mouse brain. This transgenic line is a powerful tool for studying the expression of ABCG2 in many tissues and for performing functional studies in different experimental settings.
Orford, Michael; Mean, Richard; Lapathitis, George; Genethliou, Nicholas; Panayiotou, Elena; Panayi, Helen [The Cyprus Institute of Neurology and Genetics, Airport Avenue, No. 6, Agios Dometios 2370, Nicosia (Cyprus)] [The Cyprus Institute of Neurology and Genetics, Airport Avenue, No. 6, Agios Dometios 2370, Nicosia (Cyprus); Malas, Stavros, E-mail: firstname.lastname@example.com [The Cyprus Institute of Neurology and Genetics, Airport Avenue, No. 6, Agios Dometios 2370, Nicosia (Cyprus) [The Cyprus Institute of Neurology and Genetics, Airport Avenue, No. 6, Agios Dometios 2370, Nicosia (Cyprus); Department of Biological Sciences, University of Cyprus, P.O. Box 20537, 1678 Nicosia (Cyprus)
Assessment of Fetal Cell Chimerism in Transgenic Pig Lines Generated by Sleeping Beauty Transposition
Human cells migrate between mother and fetus during pregnancy and persist in the respective host for long-term after birth. Fetal microchimerism occurs also in twins sharing a common placenta or chorion. Whether microchimerism occurs in multiparous mammals such as the domestic pig, where fetuses have separate placentas and chorions, is not well understood. Here, we assessed cell chimerism in litters of wild-type sows inseminated with semen of transposon transgenic boars. Segregation of three independent monomeric transposons ensured an excess of transgenic over non-transgenic offspring in every litter. Transgenic siblings (n?=?35) showed robust ubiquitous expression of the reporter transposon encoding a fluorescent protein, and provided an unique resource to assess a potential cell trafficking to non-transgenic littermates (n?=?7) or mothers (n?=?4). Sensitive flow cytometry, fluorescence microscopy, and real-time PCR provided no evidence for microchimerism in porcine littermates, or piglets and their mothers in both blood and solid organs. These data indicate that the epitheliochorial structure of the porcine placenta effectively prevents cellular exchange during gestation.
Garrels, Wiebke; Holler, Stephanie; Taylor, Ulrike; Herrmann, Doris; Niemann, Heiner; Ivics, Zoltan; Kues, Wilfried A.
Characterization of Multiple Bistratified Retinal Ganglion Cells in A Purkinje Cell Protein 2-Cre Transgenic Mouse Line
Retinal ganglion cells are categorized into multiple classes, including multiple types of bistratified ganglion cells (BGCs). The recent use of transgenic mouse lines with specific type(s) of ganglion cells that are labeled by fluorescent markers has facilitated the morphological and physiological studies of BGCs, particularly the directional-selective BGCs. The most important benefit from using transgenic animals is the capability to perform in vivo gene manipulation. In particular, the Cre/LoxP recombination system has become a powerful tool, allowing gene deletion, over-expression, and ectopic expression in a cell type-specific and temporally controlled fashion. The key to this tool is the availability of Cre mouse lines with cell or tissue type-specific expression of Cre recombinase. In this study, we characterized the Cre-positive retinal ganglion cells in a PCP2 (Purkinje cell protein 2)-cre mouse line. We found that all of the Cre-positive retinal ganglion cells were BGCs. Based on morphological criteria, we determined that they can be grouped into five types. The On- and Off-dendrites of three of these types stratified outside of the cholinergic bands and differed from directional selective ganglion cells (DSGCs) morphologically. These cells were negative for Brn-3b and positive for both calretinin and CART retina markers. The remaining two types were identified as putative On-Off and On-DSGCs. This Cre mouse line could be useful for further studies of the molecular and functional properties of BGCs in mice.
Ivanova, Elena; Lee, Patrick; Pan, Zhuo-Hua
[Creation of transgenic sugar beet lines expressing insect pest resistance genes cry1C and cry2A].
Impact of insect pests makes a significant limitation of the sugar beet crop yield. Integration of cry-genes of Bacillus thuringiensis into plant genome is one of the promising strategies to ensure plant resistance. The aim of this work was to obtain sugar beet lines (based on the MM 1/2 line) transformed with cry2A and cry1Cgenes. We have optimized transformation protocol and direct plant let regeneration protocol from leaf explants using 1 mg/l benzylaminopurine as well as 0,25 mg/l benzylaminopurine and 0,1 mg/l indole-butyric acid. Consequently, transgenic sugar beet lines transformed with vector constructs pRD400-cry1C and pRD400-cry2A have been obtained. PCR analysis revealed integration of cry2A and cry1C into genome of transgenic lines and expression of these genes in leaf tissues was shown by reverse transcription PCR. PMID:24818505
Litvin, D I; Sivura, V V; Kurilo, V V; Oleneva, V D; Emets, A I; Blium, Ia B
Nutritional properties of tubers of conventionally bred and transgenic lines of potato resistant to necrotic strain of Potato virus Y (PVYN).
The potential effect of genetic modification on nutritional properties of potatoes transformed to improve resistance to a necrotic strain of Potato virus Y was determined in a rat experiment. Autoclaved tubers from four transgenic lines were included to a diet in the amount of 40% and compared with the conventional cv. Irga. The experiment lasted 3 weeks and special attention was paid to nutritional properties of diets, caecal metabolism and serum indices. Genetic modification of potato had no negative effect on the chemical composition and nutritional properties of tubers, ecosystem of the caecum, activity of serum enzymes and non-specific defence mechanism of the rats. Obtained results indicate that transgenic potato with improved resistance to PVY(N): line R1F (truncated gene coding for PVY(N) polymerase in sense orientation), R2P (truncated gene coding for PVY(N) polymerase in antisense orientation), and NTR1.16 (non-translated regions of PVY(N) genome in sense orientation) are substantial and nutritional equivalence to the non-transgenic cultivar. Tubers of transgenic line NTR2.27 (non-translated regions of PVY(N) genome in antisense orientation) increased the bulk of caecal digesta and the production of SCFA as compared to tubers of the conventional cultivar and the other transgenic clones. Taking into account some deviations, it seems reasonable to undertake a long-term feeding study to confirm the nutritional properties of tubers of transgenic lines. PMID:16175247
Ju?kiewicz, Jerzy; Zdu?czyk, Zenon; Fornal, J¾zef
High-efficiency vitrification protocols for cryopreservation of in vitro grown shoot tips of transgenic papaya lines
In vitro grown shoot tips of transgenic papaya lines (Carica papaya L.) were successfully cryopreserved by vitrification. Shoot tips were excised from stock shoots that were preconditioned\\u000a in vitro for 45¢50-day-old and placed on hormone-free MS medium with 0.09ĀM sucrose. After loading for 60Āmin with a mixture\\u000a of 2ĀM glycerol and 0.4ĀM sucrose at 25░C, shoot tips were dehydrated with
Shu-Fen Tsai; Shyi-Dong Yeh; Chin-Feng Chan; Song-Iuan Liaw
Derivation and characterization of embryonic stem cells lines derived from transgenic Fischer 344 and Dark Agouti rats.
Rat embryonic stem cell (ESC) lines are not widely available, and there are only 2 lines available for distribution. Here, ESC lines were derived and characterized from Fischer 344 (F344) rats that express marker transgenes either ?-galactosidase or human placental alkaline phosphatase (AP), nontransgenic F344 rats, and from Dark Agouti (DA) rats. The ESC lines were maintained in an undifferentiated state as characterized by colony morphology, expression of Oct4, Nanog, Sox-2, Cdx2, and Stella, staining for AP, and stage-specific embryonic antigen-1. Pluripotency was demonstrated in vitro by differentiation to embryoid bodies, followed by embryonic monsters. The Cdx2 expression by ESCs was unexpected and was confirmed via reverse transcriptase-polymerase chain reaction, immunocytochemistry. Pluripotency of ESCs was demonstrated in vivo by production of teratoma after an injection into F344 nontransgenic rats, and by an injection of male DA ESCs into F344 or Sprague-Dawley rat blastocysts and the generation of chimeric rats and germline contribution. ESCs from both F344 and DA contributed to chimeric rats, and one DA ESC line was proved to be germline competent. ESC sublines were created by transfection with a plasmid expressing enhanced green fluorescent protein (eGFP) under the control of a beta actin promoter and cytomegalovirus enhancer (pCX-eGFP) or by transfection with a plasmid expressing GFP under the control of a 3.1 kb portion of the rat Oct4 promoter (pN1-Oct4-GFP). In pN1-Oct4-GFP sublines, GFP gene expression and fluorescence were shown to be correlated with endogenous Oct4 gene expression. Therefore, these new ESC lines may be useful for tissue engineering and transplantation studies or for optimizing culture conditions required for self-renewal and differentiation of rat ESCs. While they made chimeric rats, further work is needed to confirm whether the transgenic F344 rat ESCs described here are germline-competent ESCs. PMID:21995453
Hong, James; He, Hong; Weiss, Mark L
Study of heat and radiation response of a malignant, melanin-producing cell line derived from C3H 10T1/2 cells transformed in culture by radiation
The mouse C3H 10T1/2 cell line was transformed to the malignant state using ionizing radiation. One of the transformed lines (R25) that was isolated, displayed some properties similar to malignant melanoma cells. The cells became dark and pigmented after prolonged time in culture and this cell line produced tumors in C3H mice. The radiation survival curve of R25 had a large shoulder which was also observed for human melanoma cell lines. R25 was more resistant to heating at 45.0 degrees C than the normal cell line. Heating at 45.0 degrees C before irradiation resulted in a reduction of the survival curve shoulder. The heat and radiation sensitivity of R25 did not appear to be related to the melanin content of these cells.
Raaphorst, G.P.; Vadasz, J.; Azzam, E.I.
Adult onset cardiac dilatation in a transgenic mouse line with Gal?1,3GalNAc ?2,3-sialyltransferase II (ST3Gal-II) transgenes: a new model for dilated cardiomyopathy
Sugar chain abnormalities in glycolipids and glycoproteins are associated with various diseases. Here, we report an adult onset cardiac dilatation in a transgenic mouse line with Gal?1,3GalNAc ?2,3-sialyltransferase II (ST3Gal-II) transgenes. The transgenic hearts at the end-stage, at around 7 months old, were enlarged, with enlarged cavities and thin, low-tensile walls, typical of dilated cardiomyopathy. Although no apparent change was found in heart gangliosides, glycosylation of heart proteins was altered. Interestingly, sugar moieties not directly related to the ST3Gal-II catalytic reaction were also changed. Significant increases in calreticulin and calnexin were observed in hearts of the transgenic mice. These results suggest that expression of ST3Gal-II transgenes induces abnormal protein glycosylation, which disorganizes the endoplasmic/sarcoplasmic reticulum quality control system and elevates the calreticulin/calnexin level, resulting in suppression of cardiac function. The transgenic mice showed 100% incidence of adult onset cardiac dilatation, suggesting great potential as a new model for dilated cardiomyopathy.
SUZUKI, Osamu; KANAI, Takao; NISHIKAWA, Toshio; YAMAMOTO, Yoshie; NOGUCHI, Akira; TAKIMOTO, Kazuhiro; KOURA, Minako; NOGUCHI, Yoko; UCHIO-YAMADA, Kozue; TSUJI, Shuichi; MATSUDA, Junichiro
All-trans retinoic acid inhibits KIT activity and induces apoptosis in gastrointestinal stromal tumor GIST-T1 cell line by affecting on the expression of survivin and Bax protein
Background Imatinib, a selective tyrosine kinase inhibitor, has been used as a standard first-line therapy for irresectable and metastasized gastrointestinal stromal tumor (GIST) patients. Unfortunately, most patients responding to imatinib will eventually exhibit imatinib-resistance, the cause of which is not fully understood. The serious clinical problem of imatinib-resistance demands alternative therapeutic strategy. This study was conducted to investigate the effect of all-trans retinoic acid (ATRA) on GIST cell lines. Methods Cell proliferation was determined by trypan blue dye exclusion test. Western blot analysis was performed to test the expression of activated KIT, its downstream proteins, and apoptosis associated proteins. The cytotoxic interactions of imatinib with ATRA were evaluated using the isobologram of Steel and Peckham. Results and conclusion In this work, for the first time we have demonstrated that ATRA affected on cell proliferation of GIST-T1 and GIST-882 cell line through inhibition of cell growth in a dose dependent manner and induced apoptosis. High dose of ATRA induced morphologic change in GIST-T1 cells, rounded-up cells, and activated the caspase-3 protein. In further examination, we found that the ATRA-induced apoptosis in GIST-T1 cells was accompanied by the down-regulated expression of survivin and up-regulated expression of Bax protein. Moreover, ATRA suppressed the activity of KIT protein in GIST-T1 cells and its downstream signal, AKT activity, but not MAPK activity. We also have demonstrated that combination of ATRA with imatinib showed additive effect by isobologram, suggesting that the combination of ATRA and imatinib may be a novel potential therapeutic option for GIST treatment. Furthermore, the scracht assay result suggested that ATRA was a potential reagent to prevent the invasion or metastasis of GIST cells.
Transgene flow to hybrid rice and its male-sterile lines
Gene flow from genetically modified (GM) crops to the same species or wild relatives is a major concern in risk assessment.\\u000a Transgenic rice with insect and\\/or disease resistance, herbicide, salt and\\/or drought tolerance and improved quality has been\\u000a successfully developed. However, data on rice gene flow from environmental risk assessment studies are currently insufficient\\u000a for the large-scale commercialization of GM
Shirong Jia; Feng Wang; Lei Shi; Qianhua Yuan; Wuge Liu; Yilong Liao; Shuguang Li; Wujun Jin; Huipu Peng
Generation of a mouse line harboring a Bi-transgene expressing luciferase and tamoxifen-activatable creER(T2) recombinase in cartilage.
We have used an aggrecan gene enhancer to generate a transgenic murine line (Acan-CreER-Ires-Luc) expressing firefly luciferase and tamoxifen activatable Cre recombinase (Cre-ER(T2) ). The expression and efficiency of the inducible Cre recombinase activity were tested in double transgenic mice created by crossing the Acan-CreER-Ires-Luc line with a Rosa26-lacZ reporter mouse. The expression pattern of the transgene of our line was restricted to cartilage from embryonic to adult stages. ?-galactosidase staining was observed in growth plate, articular cartilage, as well as fibrocartilage of meniscus, trachea, and intervertebral discs. Similar staining was observed in a previously described Agc1 (tm(IRES-creERT2)) murine line. The presence of luciferase in our transgene allows the visualization of the transgene expression in live animals. Weekly measurements from 2 to 8 weeks of age showed a reduction in luminescence in knee joints between 2 and 4 weeks of age, but stabilization thereafter. Following the surgical induction of osteoarthritis at 12 weeks of age, the level of luminescence remained the same in the knee joints for 8 weeks. This Acan-CreER-Ires-Luc murine line allows indirect monitoring of the transcriptional activity of the Acan gene via expression of luciferase, while the inducible Cre recombinase activity facilitates studies involving gain or loss of gene expression in cartilage. PMID:24339176
Lo Cascio, Leandro; Liu, Ke; Nakamura, Hiro; Chu, Grace; Lim, Ngee Han; Chanalaris, Anastasios; Saklatvala, Jeremy; Nagase, Hideaki; Bou-Gharios, George
Production of Immortalized Distal Respiratory Epithelial Cell Lines from Surfactant Protien C\\/Simian Virus 40 Large Tumor Antigen Transgenic Mice
Murine lung epithelial (MLE) cell lines representing the distal bronchiolar and alveolar epithelium were produced from lung tumors generated in transgenic mice harboring the viral oncogene simian virus 40 (SV40) large tumor antigen under transcriptional control of a promoter region from the human surfactant protein C (SP-C) gene. The cell lines exhibited rapid growth, lack of contact inhibition, and an
Kathryn A. Wikenheiser; Diane K. Vorbroker; Ward R. Rice; Jean C. Clark; Cindy J. Bachurski; Herbert K. Oie; Jeffrey A. Whitsett
c-RET Molecule in Malignant Melanoma from Oncogenic RET-Carrying Transgenic Mice and Human Cell Lines
Malignant melanoma is one of the most aggressive cancers and its incidence worldwide has been increasing at a greater rate than that of any other cancer. We previously reported that constitutively activated RFP-RET-carrying transgenic mice (RET-mice) spontaneously develop malignant melanoma. In this study, we showed that expression levels of intrinsic c-Ret, glial cell line-derived neurotrophic factor (Gdnf) and Gdnf receptor alpha 1 (Gfra1) transcripts in malignant melanomas from RET-transgenic mice were significantly upregulated compared with those in benign melanocytic tumors. These results suggest that not only introduced oncogenic RET but also intrinsic c-Ret/Gdnf are involved in murine melanomagenesis in RET-mice. We then showed that c-RET and GDNF transcript expression levels in human malignant melanoma cell lines (HM3KO and MNT-1) were higher than those in primary cultured normal human epithelial melanocytes (NHEM), while GFRa1 transcript expression levels were comparable among NHEM, HM3KO and MNT-1. We next showed c-RET and GFRa1 protein expression in HM3KO cells and GDNF-mediated increased levels of their phosphorylated c-RET tyrosine kinase and signal transduction molecules (ERK and AKT) sited potentially downstream of c-RET. Taken together with the finding of augmented proliferation of HM3KO cells after GDNF stimulation, our results suggest that GDNF-mediated c-RET kinase activation is associated with the pathogenesis of malignant melanoma.
Takeda, Kozue; Iida, Machiko; Kumasaka, Mayuko; Matsumoto, Yoshinari; Kato, Masashi
[Analysis of mosaic expression of the nptII gene in transgenic tobacco plant lines contrasting in mosaicism].
Differences in the frequency of occurrence of plants with the mosaic phenotype between the Nu5 and Nu6 lines of transgenic tobacco (Nicotiana tabacum) plants remained irrespective of the allelic state of the nptII marker gene. Transition of the nptII gene from the hemizygous state (T3) to the homozygous state (T4) was accompanied by an increase in the frequency of mosaics in both lines. Transition from the homozygous state (T4) into the hemizygous state (F1) resulted in a further increase in the frequency of mosaic plants in the Nu5 line, whereas in the Nu6 line this parameter remained at a high level. Hypermethylation of the pMAS promoter in plants of both lines, as well as differences in the degree of methylation of cytosines in the 5'-region of the coding part of a truncated nptII gene copy between the Nu5 and Nu6 lines, pointed to epigenetic regulation of the mosaic expression of the nptII gene. PMID:23297483
Loginova, D B; Men'shanov, P N; De?neko, E V
The ability to introduce foreign genes into the germ line and the successful expression of the inserted gene in the organism have allowed the genetic manipulation of animals on an unprecedented scale. The information gained from the use of the transgenic technology is relevant to almost any aspect of modern biology including developmental gene regulation, the action of oncogenes, the
Rudolf Jaenisch
Establishment of Transgenic Lines for Jumpstarter Method Using a Composite Transposon Vector in the Ladybird Beetle, Harmonia axyridis
In this post-genomic era, genome-wide functional analysis is indispensable. The recent development of RNA interference techniques has enabled researchers to easily analyze gene function even in non-model organisms. On the other hand, little progress has been made in the identification and functional analyses of cis-regulatory elements in non-model organisms. In order to develop experimental platform for identification and analyses of cis-regulatory elements in a non-model organism, in this case, the ladybird beetle, Harmonia axyridis, we established transgenic transposon-tagged lines using a novel composite vector. This vector enables the generation of two types of insertion products (jumpstarter and mutator). The jumpstarter portion carries a transposase gene, while the mutator segment carries a reporter gene for detecting enhancers. The full-composite element is flanked by functional termini (required for movement); however, the mutator region has an extra terminus making it possible for the mutator to remobilize on its own, thus leaving an immobile jumpstarter element behind. Each insertion type is stable on its own, but once crossed, jumpstarters can remobilize mutators. After crossing a jumpstarter and mutator line, all tested G2 females gave rise to at least one new insertion line in the next generation. This jumping rate is equivalent to the P-element-mediated jumpstarter method in Drosophila. These established transgenic lines will offer us the ideal experimental materials for the effective screening and identification of enhancers in this species. In addition, this jumpstarter method has the potential to be as effective in other non-model insect species and thus applicable to any organism.
Kuwayama, Hisashi; Gotoh, Hiroki; Konishi, Yusuke; Nishikawa, Hideto; Yaginuma, Toshinobu; Niimi, Teruyuki
Cotton GalT1 Encoding a Putative Glycosyltransferase Is Involved in Regulation of Cell Wall Pectin Biosynthesis during Plant Development
Arabinogalactan proteins (AGPs), are a group of highly glycosylated proteins that are found throughout the plant kingdom. To date, glycosyltransferases that glycosylate AGP backbone have remained largely unknown. In this study, a gene (GhGalT1) encoding a putative ?-1,3-galactosyltransferase (GalT) was identified in cotton. GhGalT1, belonging to CAZy GT31 family, is the type II membrane protein that contains an N-terminal transmembrane domain and a C-terminal galactosyltransferase functional domain. A subcellular localization assay demonstrated that GhGalT1 was localized in the Golgi apparatus. RT-PCR analysis revealed that GhGalT1 was expressed at relatively high levels in hypocotyls, roots, fibers and ovules. Overexpression of GhGalT1 in Arabidopsis promoted plant growth and metabolism. The transgenic seedlings had much longer primary roots, higher chlorophyll content, higher photosynthetic efficiency, the increased biomass, and the enhanced tolerance to exogenous D-arabinose and D-galactose. In addition, gas chromatography (GC) analysis of monosaccharide composition of cell wall fractions showed that pectin was changed in the transgenic plants, compared with that of wild type. Three genes (GAUT8, GAUT9 and xgd1) involved in pectin biosynthesis were dramatically up-regulated in the transgenic lines. These data suggested that GhGalT1 may be involved in regulation of pectin biosynthesis required for plant development.
Qin, Li-Xia; Rao, Yue; Li, Long; Huang, Jun-Feng; Xu, Wen-Liang; Li, Xue-Bao
Transgenic resistance to cucumber mosaic virus in tomato: blocking of long-distance movement of the virus in lines harboring a defective viral replicase gene.
ABSTRACT Tomato breeding lines were transformed with a defective replicase gene from RNA 2 of cucumber mosaic virus (CMV). A total of 63 transformants from five tomato genotypes were evaluated for resistance to CMV strains. The responses of R1 transgenic offspring fit into three categories: fully susceptible lines (44%), fully resistant lines (8%), and an intermediate-type mixture of susceptible and resistant seedlings in variable proportions (48%). Further characterization of the response of two highly resistant lines was performed by mechanical inoculation, aphid transmission, or grafting experiments. No virus was detected in noninoculated leaves from these lines, although a low level of virus accumulated initially in the inoculated leaf. The homozygous R2 plants and further generations that were evaluated (up to R5) showed resistance to the Fny-CMV strain, two Israeli isolates tentatively classified as subgroup IA, and K-CMV (a representative of subgroup IB). These lines were partially resistant to LS-CMV (a representative of subgroup II) when a high-virus-titer inoculum was used. Expression of the viral transgene was verified in these lines; however, the expected translation product was not detectable. In grafting experiments, we demonstrated that CMV virions were blocked in their ability to move from infected rootstocks of nontransformed tomato or tobacco into the transgenic scions. Interestingly, virions could not move through a transgenic intersection into the upper scion. These results provide an additional indication that replicase-mediated resistance affects long-distance movement. PMID:18944823
Gal-On, A; Wolf, D; Wang, Y; Faure, J E; Pilowsky, M; Zelcer, A
Characterization of Tu-2449, a glioma cell line derived from a spontaneous tumor in GFAP-v-src-transgenic mice: comparison with established murine glioma cell lines.
Glial fibrillary acidic protein (GFAP)-v-src transgenic mice develop spontaneous gliomas with a high incidence of malignant progression. We characterize the first glial cell line derived from v-src transgenic mice, Tu-2449 in comparison with a virally induced murine glioma, SRB-10, and a spontaneous murine glioma, P497. Doubling times were lowest, as clonogenicity in soft agar was highest for Tu-2449, and to a lesser degree, Tu-2449 cells formed spheroids and showed migratory behaviour and invaded fetal rat brain aggregates. BCL-2 and BAX expression were lower in Tu-2449 and P497 than in SRB-10 cells. Only Tu-2449 cells accumulated p53 protein in response to genotoxic stress. Tu-2449 and SRB-10 cells that showed low CD95 expression were resistant to CD95 ligand (CD95L)-induced apoptosis unless coexposed to CD95L and inhibitors of RNA or protein synthesis. A chemosensitivity profile revealed Tu-2449 to be rather chemoresistant. Tu-2449 thus displays growth characteristics and patterns of resistance to apoptosis similar to those of other mouse and human glioma cell lines and may therefore become a valuable tool to evaluate new therapies for malignant gliomas in vitro and in vivo. PMID:10493969
Pohl, U; Wick, W; Weissenberger, J; Steinbach, J P; Dichgans, J; Aguzzi, A; Weller, M
Development of transgenic lines of Eimeria tenella expressing M2e-enhanced yellow fluorescent protein (M2e-EYFP).
Eimeria parasites are obligate intracellular apicomplexan protists that can cause coccidiosis, resulting in substantial economic losses in the poultry industry annually. As the component of anticoccidial vaccines, seven Eimeria spp. of chickens are characterized with potent immunogenicity. Whether genetically modified Eimeria spp. maintains this property or not needs to be verified. In this study, two identical transgenic lines of Eimeria tenella were developed by virtue of single sporocyst isolation from a stably transfected population expressing fused protein of M2 ectodomain of avian influenza virus (M2e) and enhanced yellow fluorescent protein (EYFP). The chromosomal integration and expression of M2e-EYFP were confirmed by Southern blot, plasmid rescue and Western blot analysis. We found that the reproduction of transgenic parasites was higher than that of the parental strain. Chickens challenged with wild type E. tenella after immunization with 200 oocysts of transgenic parasites had similar performance compared to those in non-immunized and non-challenged group. In another trial, the performance of transgenic parasite-immunized birds was also comparable to that of the Decoquinate Premix-treated chickens. These results suggest that this transgenic line of E. tenella is capable of inducing potent protection against homologous challenge as a live anticoccidial vaccine. Taking together, our study indicates that transgenic eimerian parasites have the potential to be developed as a vaccine vehicle for animal use in the future. PMID:23298569
Liu, Xianyong; Zou, Jun; Yin, Guangwen; Su, Huali; Huang, Xiaoxi; Li, Jianan; Xie, Li; Cao, Yingqiong; Cui, Yujuan; Suo, Xun
Fluorescence-Tagged Transgenic Lines Reveal Genetic Defects in Pollen Growth--Application to the Eif3 Complex
Background Mutations in several subunits of eukaryotic translation initiation factor 3 (eIF3) cause male transmission defects in Arabidopsis thaliana. To identify the stage of pollen development at which eIF3 becomes essential it is desirable to examine viable pollen and distinguish mutant from wild type. To accomplish this we have developed a broadly applicable method to track mutant alleles that are not already tagged by a visible marker gene through the male lineage of Arabidopsis. Methodology/Principal Findings Fluorescence tagged lines (FTLs) harbor a transgenic fluorescent protein gene (XFP) expressed by the pollen-specific LAT52 promoter at a defined chromosomal position. In the existing collection of FTLs there are enough XFP marker genes to track nearly every nuclear gene by virtue of its genetic linkage to a transgenic marker gene. Using FTLs in a quartet mutant, which yields mature pollen tetrads, we determined that the pollen transmission defect of the eif3h-1 allele is due to a combination of reduced pollen germination and reduced pollen tube elongation. We also detected reduced pollen germination for eif3e. However, neither eif3h nor eif3e, unlike other known gametophytic mutations, measurably disrupted the early stages of pollen maturation. Conclusion/Significance eIF3h and eIF3e both become essential during pollen germination, a stage of vigorous translation of newly transcribed mRNAs. These data delimit the end of the developmental window during which paternal rescue is still possible. Moreover, the FTL collection of mapped fluorescent protein transgenes represents an attractive resource for elucidating the pollen development phenotypes of any fine-mapped mutation in Arabidopsis.
Roy, Bijoyita; Copenhaver, Gregory P.; von Arnim, Albrecht G.
Transgenic maize lines with cell-type specific expression of fluorescent proteins in plastids.
Plastid number and morphology vary dramatically between cell types and at different developmental stages. Furthermore, in C4 plants such as maize, chloroplast ultrastructure and biochemical functions are specialized in mesophyll and bundle sheath cells, which differentiate acropetally from the proplastid form in the leaf base. To develop visible markers for maize plastids, we have created a series of stable transgenics expressing fluorescent proteins fused to either the maize ubiquitin promoter, the mesophyll-specific phosphoenolpyruvate carboxylase (PepC) promoter, or the bundle sheath-specific Rubisco small subunit 1 (RbcS) promoter. Multiple independent events were examined and revealed that maize codon-optimized versions of YFP and GFP were particularly well expressed, and that expression was stably inherited. Plants carrying PepC promoter constructs exhibit YFP expression in mesophyll plastids and the RbcS promoter mediated expression in bundle sheath plastids. The PepC and RbcS promoter fusions also proved useful for identifying plastids in organs such as epidermis, silks, roots and trichomes. These tools will inform future plastid-related studies of wild-type and mutant maize plants and provide material from which different plastid types may be isolated. PMID:20051034
Sattarzadeh, Amir; Fuller, Jonathan; Moguel, Salvador; Wostrikoff, Katia; Sato, Shirley; Covshoff, Sarah; Clemente, Tom; Hanson, Maureen; Stern, David B
Down-Regulation of Transmembrane Carbonic Anhydrases in Renal Cell Carcinoma Cell Lines by Wild-Type von Hippel-Lindau Transgenes
To discover genes involved in von Hippel-Lindau (VHL)-mediated carcinogenesis, we used renal cell carcinoma cell lines stably transfected with wild-type VHL-expressing transgenes. Large-scale RNA differential display technology applied to these cell lines identified several differentially expressed genes, including an alpha carbonic anhydrase gene, termed CA12. The deduced protein sequence was classified as a one-pass transmembrane CA possessing an apparently intact
Sergey V. Ivanov; Igor Kuzmin; Ming-Hui Wei; Svetlana Pack; Laura Geil; Bruce E. Johnson; Eric J. Stanbridge; Michael I. Lerman
Isolation and characterization of a mesenchymal cell line that differentiates into osteoblasts in response to BMP2 from calvariae of GFP transgenic mice
We established the clonal mesenchymal cell line, GFP-C3 (C3), which differentiates into osteoblasts in response to BMP-2 from calvariae of newborn green fluorescence protein (GFP) transgenic mice. This cell line cultured with control medium expressed low levels of alkaline phosphatase (ALP) activity and osterix mRNA and undetectable ALP and osteocalcin mRNA. Incubation of these cells with rhBMP-2 increased ALP activity
A Kadowaki; T Tsukazaki; K Hirata; Y Shibata; Y Okubo; K Bessho; T Komori; N Yoshida; A Yamaguchi
Transgenic rice plants expressing cry1Ia5 gene are resistant to stem borer (Chilo agamemnon).
The stem borer, Chilo agamemnon Bles., is the most serious insect pest in rice fields of the Egyptian Nile Delta. To induce rice plant resistance to Chilo agamemnon, the cry1Ia5 gene was introduced to rice plants (Oryza sativa L.). The integration of the cry1Ia5 gene into the plant genome was confirmed using PCR and Southern blot analyses. The obtained plantlets were transferred to the greenhouse until seeds were collected. Northern blot analysis of the T1 plants confirmed the expression of the cry1Ia5 gene. The insecticidal activity of the transgenic plants against the rice stem borer Chilo agamemnon were tested. The third larval instars were fed on stem cuts from three transgenic lines (L1, L2 and L3) as well as cuts from the control (gfp-transgenic) plants for one week and the mortality percentage was daily recorded. Transgenic line-3 showed the highest mortality percentage after one day (50%) followed by L2 (25%) then L1 (0%). Two days post treatment the mortality percentage increased to 70, 45 and 25% for transgenic lines 1, 2 and 3 respectively. Mortality of 100% was recorded four days post treatment, while those fed on the gfp-transgenic rice (control) showed 0% mortality. Thus, transgenic plants showed high resistance to stem borers and can serve as a novel genetic resource in breeding programs. Transgenic plants expressing BT protein were normal in phenotype with as good seed setting as the nontransgenic control plants. PMID:21844686
Moghaieb, Reda E A
Analyses of single-copy Arabidopsis T-DNA-transformed lines show that the presence of vector backbone sequences, short inverted repeats and DNA methylation is not sufficient or necessary for the induction of transgene silencing
In genetically transformed plants, transgene silen- cing has been correlated with multiple and complex insertions of foreign DNA, e.g. T-DNA and vector backbone sequences. Occasionally, single-copy transgenes also suffer transgene silencing. We have compared integration patterns and T-DNA\\/plant DNA junctions in a collection of 37 single-copy T-DNA-transformed Arabidopsis lines, of which 13 displayed silencing. Vector sequences were found integrated in
Trine J. Meza; Biljana Stangeland; Inderjit S. Mercy; Magne Skarn; Dag A. Nymoen; Anita Berg; Melinka A. Butenko; Anne-Mari Hakelien; Camilla Haslekas; Leonardo A. Meza-Zepeda; Reidunn B. Aalen
Transgenic indica rice lines, expressing Brassica juncea Nonexpressor of pathogenesis-related genes 1 (BjNPR1), exhibit enhanced resistance to major pathogens.
Brassica juncea Nonexpressor of pathogenesis-related genes 1 (BjNPR1) has been introduced into commercial indica rice varieties by Agrobacterium-mediated genetic transformation. Transgenic rice plants were regenerated from the phosphinothricin-resistant calli obtained after co-cultivation with Agrobacterium strain LBA4404 harbouring Ti plasmid pSB111-bar-BjNPR1. Molecular analyses confirmed the stable integration and expression of BjNPR1 in various transgenic rice lines. Transgenes NPR1 and bar were stably inherited and disclosed co-segregation in subsequent generations in a Mendelian fashion. Homozygous transgenic rice lines expressing BjNPR1 protein displayed enhanced resistance to rice blast, sheath blight and bacterial leaf blight diseases. Rice transformants with higher levels of NPR1 revealed notable increases in plant height, panicle length, flag-leaf length, number of seeds/panicle and seed yield/plant as compared to the untransformed plants. The overall results amply demonstrate the profound impact of BjNPR1 in imparting resistance against major pathogens of rice. The multipotent BjNPR1, as such, seems promising as a prime candidate gene to fortify crop plants with durable resistance against various pathogens. PMID:23664883
Sadumpati, Vijayakumar; Kalambur, Muralidharan; Vudem, Dashavantha Reddy; Kirti, Pulugurtha Bharadwaja; Khareedu, Venkateswara Rao
Detection of feral GT73 transgenic oilseed rape (Brassica napus) along railway lines on entry routes to oilseed factories in Switzerland.
To obtain a reference status prior to cultivation of genetically modified oilseed rape (OSR, Brassica napus L.) in Switzerland, the occurrence of feral OSR was monitored along transportation routes and at processing sites. The focus was set on the detection of (transgenic) OSR along railway lines from the Swiss borders with Italy and France to the respective oilseed processing factories in Southern and Northern Switzerland (Ticino and region of Basel). A monitoring concept was developed to identify sites of largest risk of escape of genetically modified plants into the environment in Switzerland. Transport spillage of OSR seeds from railway goods cars particularly at risk hot spots such as switch yards and (un)loading points but also incidental and continuous spillage were considered. All OSR plants, including their hybridization partners which were collected at the respective monitoring sites were analyzed for the presence of transgenes by real-time PCR. On sampling lengths each of 4.2 and 5.7Ākm, respectively, 461 and 1,574 plants were sampled in Ticino and the region of Basel. OSR plants were found most frequently along the routes to the oilseed facilities, and in larger amounts on risk hot spots compared to sites of random sampling. At three locations in both monitored regions, transgenic B. napus line GT73 carrying the glyphosate resistance transgenes gox and CP4 epsps were detected (Ticino, 22 plants; in the region of Basel, 159). PMID:23917737
Hecht, Mirco; Oehen, Bernadette; Schulze, J³rg; Brodmann, Peter; Bagutti, Claudia
Establishment of a preadipocyte cell line derived from mature adipocytes of GFP transgenic mice and formation of adipose tissue.
We established a preadipocyte cell line from mature adipocytes obtained from subcutaneous fat tissue of green fluorescent protein (GFP) transgenic mice. The floating top layer, containing mature adipocytes, was isolated from subcutaneous fat tissue by collagenase digestion and filtration. Fluorescence-activated cell sorting and microscopic analysis revealed that the floating cell fraction comprised a highly homogeneous adipocyte population with no adipose stromal-vascular cells. Isolated mature adipocytes dedifferentiated into fibroblast-like cells and actively proliferated in ceiling culture. In vitro studies showed that the cells could redifferentiate into mature adipocytes in an identical way to 3T3-L1 preadipocytes. No changes in the differentiation pattern were observed during the propagation of our cells. They were successfully maintained and differentiated for at least 22 passages. We named these cells dedifferentiated fat (DFAT-GFP) cells. When DFAT-GFP cells were implanted subcutaneously into C57BL/6N mice, they developed highly vascularized fat pads that morphologically resembled normal subcutaneous adipose tissue and consisted of GFP-positive cells; however, implanted 3T3-L1 cells did not have such an effect on the mice. We conclude that DFAT-GFP cells provide a model that should enable us to study the mechanisms of adipocyte differentiation and adipose tissue formation in vivo and in vitro. PMID:18386066
Nobusue, Hiroyuki; Endo, Tsuyoshi; Kano, Koichiro
Generation of hermaphrodite transgenic papaya lines with virus resistance via transformation of somatic embryos derived from adventitious roots of in vitro shoots.
Papaya production is seriously limited by Papaya ringspot virus (PRSV) worldwide and Papaya leaf-distortion mosaic virus (PLDMV) in Eastern Asia. An efficient transformation method for developing papaya lines with transgenic resistance to these viruses and commercially desirable traits, such as hermaphroditism, is crucial to shorten the breeding program for this fruit crop. In this investigation, an untranslatable chimeric construct pYP08 containing truncated PRSV coat protein (CP) and PLDMV CP genes coupled with the 3' untranslational region of PLDMV, was generated. Root segments from different portions of adventitious roots of in vitro multiple shoots of hermaphroditic plants of papaya cultivars 'Tainung No. 2', 'Sunrise', and 'Thailand' were cultured on induction medium for regeneration into somatic embryos. The highest frequency of somatic embryogenesis was from the root-tip segments of adventitious roots developed 2-4 weeks after rooting in perlite medium. After proliferation, embryogenic tissues derived from somatic embryos were wounded in liquid-phase by carborundum and transformed by Agrobacterium carrying pYP08. Similarly, another construct pBG-PLDMVstop containing untranslatable CP gene of PLDMV was also transferred to 'Sunrise' and 'Thailand', the parental cultivars of 'Tainung No. 2'. Among 107 transgenic lines regenerated from 349 root-tip segments, nine lines of Tainung No. 2 carrying YP08 were highly resistant to PRSV and PLDMV, and 9 lines (8 'Sunrise' and 1 'Thailand') carrying PLDMV CP highly resistant to PLDMV, by a mechanism of post-transcriptional gene silencing. The hermaphroditic characteristics of the transgenic lines were confirmed by PCR with sex-linked primers and phenotypes of flower and fruit. Our approach has generated transgenic resistance to both PRSV and PLDMV with commercially desirable characters and can significantly shorten the time-consuming breeding programs for the generation of elite cultivars of papaya hybrids. PMID:19943109
Kung, Yi-Jung; Yu, Tsong-Ann; Huang, Chiung-Huei; Wang, Hui-Chin; Wang, Shin-Lan; Yeh, Shyi-Dong
Generation of transgenic watermelon resistant to Zucchini yellow mosaic virus and Papaya ringspot virus type W.
Zucchini yellow mosaic virus (ZYMV) and Papaya ringspot virus type W (PRSV W) are major limiting factors for production of watermelon worldwide. For the effective control of these two viruses by transgenic resistance, an untranslatable chimeric construct containing truncated ZYMV coat protein (CP) and PRSV W CP genes was transferred to commercial watermelon cultivars by Agrobacterium-mediated transformation. Using our protocol, a total of 27 putative transgenic lines were obtained from three cultivars of 'Feeling' (23 lines), 'China baby' (3 lines), and 'Quality' (1 line). PCR and Southern blot analyses confirmed that the chimeric construct was incorporated into the genomic DNA of the transformants. Greenhouse evaluation of the selected ten transgenic lines of 'Feeling' cultivar revealed that two immune lines conferred complete resistance to ZYMV and PRSV W, from which virus accumulation were not detected by Western blotting 4 weeks after inoculation. The transgenic transcript was not detected, but small interfering RNA (siRNA) was readily detected from the two immune lines and T(1) progeny of line ZW 10 before inoculation, indicating that RNA-mediated post-transcriptional gene silencing (PTGS) is the underlying mechanism for the double-virus resistance. The segregation ratio of T(1) progeny of the immune line ZW10 indicated that the single inserted transgene is nuclearly inherited and associated with the phenotype of double-virus resistance as a dominant trait. The transgenic lines derived from the commercial watermelon cultivars have great potential for control of the two important viruses and can be implemented directly without further breeding. PMID:21079966
Yu, Tsong-Ann; Chiang, Chu-Hui; Wu, Hui-Wen; Li, Chin-Mei; Yang, Ching-Fu; Chen, Jun-Han; Chen, Yu-Wen; Yeh, Shyi-Dong
Direct visualization of cell movement in the embryonic olfactory bulb using green fluorescent protein transgenic mice: evidence for rapid tangential migration of neural cell precursors
We analyzed motile behavior of neuronal precursor cells in the intact olfactory bulbs (OBs) using transgenic mice expressing GFP under the control of T?1 tubulin promoter. In the olfactory bulbs at the embryonic days 12.5¢14.5, a large number of immature neurons expressed GFP in this transgenic line. Embryonic OBs were maintained in an organ culture system and the migratory behavior
Kazuhiro Yamamoto; Masahiro Yamaguchi; Shigeo Okabe
Investigation of rice transgene flow in compass sectors by using male sterile line as a pollen detector
Rice is the most important staple food in the world. The rapid development of transgenic rice and its future commercialization\\u000a have raised concerns regarding transgene flow and its potential environmental risk. It is known that rice is a self-pollinated\\u000a crop; the outcrossing rate between common cultivars is generally less than 1%. In order to improve the detection sensitivity\\u000a of rice
Q. H. Yuan; L. Shi; F. Wang; B. Cao; Q. Qian; X. M. Lei; Y. L. Liao; W. G. Liu; L. Cheng; S. R. Jia
Generation and phenotypic analysis of a transgenic line of rabbits secreting active recombinant human erythropoietin in the milk.
Production of recombinant human erythropoietin (rhEPO) for therapeutic purposes relies on its expression in selected clones of transfected mammalian cells. Alternatively, this glycoprotein can be produced by targeted secretion into the body fluid of transgenic mammals. Here, we report on the generation of a transgenic rabbits producing rhEPO in the lactating mammary gland. Transgenic individuals are viable, fertile and transmit the rhEPO gene to the offspring. Northern blot data indicated that the expression of the transgene in the mammary gland is controlled by whey acidic protien (WAP) regulatory sequences during the period of lactation. While the hybridization with total RNA revealed the expression only in the lactating mammary gland, the highly sensitive combinatory approach using RT-PCR/hybridization technique detected a minor ectopic expression. The level of rhEPO secretion in the founder female, measured in the period of lactation, varied in the range of 60-178 and 60-162 mIU/ml in the milk and blood plasma, respectively. Biological activity of the milk rhEPO was confirmed by a standard [3H]-thymidine incorporation test. Thus, we describe the model of a rhEPO-transgenic rabbit, valuable for studies of rhEPO glycosylation and function, which can be useful for the development of transgenic approaches designed for the preparation of recombinant proteins by alternative biopharmaceutical production. PMID:15587272
Mikus, Tomßs; Poplstein, Martin; Sedlßkovß, Jirina; Landa, VladimĒr; JenĒkova, Gabriela; Trefil, Pavel; Lidick², Jan; Mal², Petr
Development of a Transgenic Plasmodium berghei Line (Pbpfpkg) Expressing the P. falciparum cGMP-Dependent Protein Kinase, a Novel Antimalarial Drug Target
With the inevitable selection of resistance to antimalarial drugs in treated populations, there is a need for new medicines to enter the clinic and new targets to progress through the drug discovery pipeline. In this study we set out to develop a transgenic rodent model for testing inhibitors of the Plasmodium falciparum cyclic GMP-dependent kinase in vivo. A model was needed that would allow us to investigate whether differences in amino acid sequence of this enzyme between species influences in vivo efficacy. Here we report the successful development of a transgenic P. berghei line in which the cyclic GMP-dependent protein kinase (PKG) was replaced by the P. falciparum orthologue. We demonstrate that the P. falciparum orthologue was able to functionally complement the endogenous P. berghei pkg gene throughout blood stage development and early sexual development. However, subsequent development in the mosquito was severely compromised. We show that this is due to a defect in the female lineage of the transgenic by using genetic crosses with both male and female deficient P. berghei lines. This defect could be due to expression of a female-specific target in the mosquito stages of P. berghei that cannot be phosphorylated by the P. falciparum kinase. Using a previously reported anti-coccidial inhibitor of the cyclic GMP-dependent protein kinase, we show no difference in in vivo efficacy between the transgenic and control P. berghei lines. This in vivo model will be useful for screening future generations of cyclic GMP-dependent protein kinase inhibitors and allowing us to overcome any species-specific differences in the enzyme primary sequence that would influence in vivo efficacy in the rodent model. The approach will also be applicable to in vivo testing of other antimalarial compounds where the target is known.
Tewari, Rita; Patzewitz, Eva-Maria; Poulin, Benoit; Stewart, Lindsay; Baker, David A.
Agronomic performance and transcriptional analysis of carotenoid biosynthesis in fruits of transgenic HighCaro and control tomato lines under field conditions.
Genetic manipulation of carotenoid biosynthesis in higher plants has been the objective of a number of biotechnology programs, e.g. the Golden Rice Program. However, tomato (Solanum lycopersicum L.), which naturally accumulates lycopene in fruits, has attracted the attention of many groups who have manipulated it to increase or diversify carotenoid accumulation. One of the most significant achievements was "HighCaro (HC)," a transgenic tomato plant constitutively expressing the tomato lycopene beta-cyclase (tLcy-b), that produces orange fruits due to the complete conversion of lycopene to beta-carotene. In this article we report the results of a field trial conducted in Metaponto (Italy) on HC and on two control genotypes to evaluate the stability of the transgenic trait and their yield performances. Transcriptional regulation of eight genes involved in carotenogenesis was assayed by quantitative real-time PCR (qRT-PCR) analysis on fruits collected at four distinct development stages. Statistical analysis results demonstrated that in field conditions the transgene maintained its ability to induce the conversion of lycopene to beta-carotene. Moreover, agronomic performances and fruit quality in the transgenic line were not impaired by this metabolic disturbance. Results of qRT-PCR analysis suggested that transcription of PSY-1, PDS and ZDS genes were developmentally regulated in both genotypes. Unexpectedly, Lcy-b expression in transgenic fruits was also developmentally regulated, despite the fact that the gene was driven by a constitutive promoter. Our data provide evidence that in photosynthetic cells a strict and aspecific mechanism controls the level of transcripts until the onset of chromoplasts differentiation, at which point a gene-specific control on transcription takes place. PMID:17096211
Giorio, Giovanni; Stigliani, Adriana Lucia; D'Ambrosio, Caterina
Synergistic effect of human CycT1 and CRM1 on HIV-1 propagation in rat T cells and macrophages
Background In vivo studies of HIV-1 pathogenesis and testing of antiviral strategies have been hampered by the lack of an immunocompetent small animal model that is highly susceptible to HIV-1 infection. Although transgenic rats that express the HIV-1 receptor complex hCD4 and hCCR5 are susceptible to infection, HIV-1 replicates very poorly in these animals. To demonstrate the molecular basis for developing a better rat model for HIV-1 infection, we evaluated the effect of human CyclinT1 (hCycT1) and CRM1 (hCRM1) on Gag p24 production in rat T cells and macrophages using both established cell lines and primary cells prepared from hCycT1/hCRM1 transgenic rats. Results Expression of hCycT1 augmented Gag production 20¢50 fold in rat T cells, but had little effect in macrophages. Expression of hCRM1 enhanced Gag production 10¢15 fold in macrophages, but only marginally in T cells. Expression of both factors synergistically enhanced p24 production to levels approximately 10¢40% of those detected in human cells. R5 viruses produced in rat T cells and macrophages were fully infectious. Conclusion The expression of both hCycT1 and hCRM1 appears to be fundamental to developing a rat model that supports robust propagation of HIV-1.
Okada, Hiroyuki; Zhang, Xianfeng; Ben Fofana, Ismael; Nagai, Mika; Suzuki, Hajime; Ohashi, Takashi; Shida, Hisatoshi
Characterization of the Frizzled10-CreERÖ Transgenic Mouse: An Inducible Cre Line for the Study of Cajal-Retzius Cell Development
Summary Cajal-Retzius cells are an enigmatic class of neurons located in the most superficial layer of the cerebral cortex, and they play an important role in cortical development. Although many studies have indicated that CR cells are involved in regulating cell migration and cortical maturation, the function of these cells is still not fully understood. Here we describe an inducible Cre mouse line in which CreERÖ is driven by the promoter for the Wnt receptor Frizzled10. Consistent with our previous studies on Frizzled10 expression and transgenic mouse lines using the Frizzled10 promoter, we found that in the developing telencephalon, Cre was mainly detected at the cortical hem, the largest source of CR cells. By crossing the Cre line to R26R reporter mice and injecting tamoxifen at different time points, we were able to detect via X-gal staining CR cells produced from the cortical hem at distinct stages during development. Thus, this transgenic Cre mouse line is a valuable tool for studying the molecular and cellular mechanisms of CR cell development.
Gu, Xiaochun; Yan, Yan; Li, Hanlin; He, Dongyang; Pleasure, Samuel J.; Zhao, Chunjie
Transgenic mouse lines expressing rat AH receptor variants - A new animal model for research on AH receptor function and dioxin toxicity mechanisms
Han/Wistar (Kuopio; H/W) rats are exceptionally resistant to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity mainly because of their mutated aryl hydrocarbon receptor (AHR) gene. In H/W rats, altered splicing of the AHR mRNA generates two AHR proteins: deletion (DEL) and insertion (INS) variants, with the INS isoform being predominantly expressed. To gain further insight into their functional properties, cDNAs of these and rat wild-type (rWT) isoform were transferred into C57BL/6J-derived mice by microinjection. The endogenous mouse AHR was eliminated by selective crossing with Ahr-null mice. A single mouse line was obtained for each of the three constructs. The AHR mRNA levels in tissues were generally close to those of C57BL/6 mice in INS and DEL mice and somewhat higher in rWT mice; in testis, however, all 3 constructs exhibited marked overexpression. The transgenic mouse lines were phenotypically normal except for increased testis weight. Induction of drug-metabolizing enzymes by TCDD occurred similarly to that in C57BL/6 mice, but there tended to be a correlation with AHR concentrations, especially in testis. In contrast to C57BL/6 mice, the transgenics did not display any major gender difference in susceptibility to the acute lethality and hepatotoxicity of TCDD; rWT mice were highly sensitive, DEL mice moderately resistant and INS mice highly resistant. Co-expression of mouse AHR and rWT resulted in augmented sensitivity to TCDD and abolished the natural resistance of female C57BL/6 mice, whereas mice co-expressing mouse AHR and INS were resistant. Thus, these transgenic mouse lines provide a novel promising tool for molecular studies on dioxin toxicity and AHR function.
Pohjanvirta, Raimo [Department of Food and Environmental Hygiene, Faculty of Veterinary Medicine, P.O. Box 66, FI-00014 University of Helsinki (Finland); National Institute for Health and Welfare, Laboratory of Toxicology, P.O. Box 95, FI-70701 Kuopio (Finland); Finnish Food Safety Authority EVIRA, Kuopio Research Unit, P.O. Box 92, FI-70701 Kuopio (Finland)], E-mail: firstname.lastname@example.com
Enhanced resistance to stripe rust disease in transgenic wheat expressing the rice chitinase gene RC24.
Stripe rust is a devastating fungal disease of wheat worldwide which is primarily caused by Puccinia striiformis f. sp tritici. Transgenic wheat (Triticum aestivum L.) expressing rice class chitinase gene RC24 were developed by particle bombardment of immature embryos and tested for resistance to Puccinia striiformis f.sp tritici. under greenhouse and field conditions. Putative transformants were selected on kanamycin-containing media. Polymease chain reaction indicated that RC24 was transferred into 17 transformants obtained from bombardment of 1,684 immature embryos. Integration of RC24 was confirmed by Southern blot with a RC24-labeled probe and expression of RC24 was verified by RT-PCR. Nine transgenic T1 lines exhibited enhanced resistance to stripe rust infection with lines XN8 and BF4 showing the highest level of resistance. Southern blot hybridization confirmed the stable inheritance of RC24 in transgenic T1 plants. Resistance to stripe rust in transgenic T2 and T3 XN8 and BF4 plants was confirmed over two consecutive years in the field. Increased yield (27-36Ā%) was recorded for transgenic T2 and T3 XN8 and BF4 plants compared to controls. These results suggest that rice class I chitinase RC24 can be used to engineer stripe rust resistance in wheat. PMID:23529204
Huang, Xuan; Wang, Jian; Du, Zhen; Zhang, Chen; Li, Lan; Xu, Ziqin
Quantitative Proteomics Analysis of Chondrogenic Differentiation of C3H10T1/2 Mesenchymal Stem Cells by iTRAQ Labeling Coupled with On-line Two-dimensional LC/MS/MS*
The chondrogenic potential of multipotent mesenchymal stem cells (MSCs) makes them a promising source for cell-based therapy of cartilage defects; however, the exact intracellular molecular mechanisms of chondrogenesis as well as self-renewal of MSCs remain largely unknown. To gain more insight into the underlying molecular mechanisms, we applied isobaric tag for relative and absolute quantitation (iTRAQ) labeling coupled with on-line two-dimensional LC/MS/MS technology to identify proteins differentially expressed in an in vitro model for chondrogenesis: chondrogenic differentiation of C3H10T1/2 cells, a murine embryonic mesenchymal cell line, was induced by micromass culture and 100 ng/ml bone morphogenetic protein 2 treatment for 6 days. A total of 1756 proteins were identified with an average false discovery rate <0.21%. Linear regression analysis of the quantitative data gave strong correlation coefficients: 0.948 and 0.923 for two replicate two-dimensional LC/MS/MS analyses and 0.881, 0.869, and 0.927 for three independent iTRAQ experiments, respectively (p < 0.0001). Among 1753 quantified proteins, 100 were significantly altered (95% confidence interval), and six of them were further validated by Western blotting. Functional categorization revealed that the 17 up-regulated proteins mainly comprised hallmarks of mature chondrocytes and enzymes participating in cartilage extracellular matrix synthesis, whereas the 83 down-regulated were predominantly involved in energy metabolism, chromatin organization, transcription, mRNA processing, signaling transduction, and cytoskeleton; except for a number of well documented proteins, the majority of these altered proteins were novel for chondrogenesis. Finally, the biological roles of BTF3l4 and fibulin-5, two novel chondrogenesis-related proteins identified in the present study, were verified in the context of chondrogenic differentiation. These data will provide valuable clues for our better understanding of the underlying mechanisms that modulate these complex biological processes and assist in the application of MSCs in cell-based therapy for cartilage regeneration.
Ji, Yu-hua; Ji, Ju-ling; Sun, Fen-yong; Zeng, Yao-ying; He, Xian-hui; Zhao, Jing-xian; Yu, Yu; Yu, Shou-he; Wu, Wei
Transgenic zebrafish line with over-expression of Hedgehog on the skin: a useful tool to screen Hedgehog-inhibiting compounds
We generated a transgenic line Tg(k18:shh:RFP) with overexpression of Sonic hedgehog in the skin epidermis. By 5Āday-post-fertilization (dpf), many epidermal lesions were\\u000a clearly observed, including a swollen yolk sac, epidermis growth malformation around the eyes and at the basement of the pectoral\\u000a fins. Skin histology revealed embryos derived from Tg(k18:shh:RFP) displayed an elevated Nuclear\\/Cytoplasmic ratio and pleomorphic nuclei compared to
Yau-Hung Chen; Yun-Hsin Wang; Tsung-Han Yu; Hsin-Ju Wu; Chiung-Wen Pai
Effects of expression of human or bovine growth hormone genes on sperm production and male reproductive performance in four lines of transgenic mice
Summary. Reproductive performance was studied in transgenic males from lines expressing and transmitting four hybrid genes: mouse metallothionein-I\\/human growth hormone (GH) (MT\\/hGH), MT\\/hGH placental variant (MT\\/hGH\\\\m=.\\\\V),MT\\/bovine GH (MT\\/bGH) and phosphoenolpyruvate carboxykinase\\/bGH (PEPCK\\/bGH). Each male was exposed to three normal females for 1 week and to three different normal females for another week. Females were examined for vaginal plugs and necropsied
A. Bartke; E. M. Naar; L. Johnson; M. R. May; M. Cecim; J. S. Yun; T. E. Wagner
[Overexpression of the glutathione S-transferase gene from Pyrus pyrifolia fruit improves tolerance to abiotic stress in transgenic tobacco plants].
Glutathione S-transferases (GSTs) are ubiquitous enzymes in animals and plants, and they are multifunctional proteins encoded by a large gene family. GSTs are involved in response to the oxidative stress including drought, salt, heavy metals, and so on. Under oxidative stress, the excessive reactive oxygen species (ROS) induce an increase in GST levels, and then the GSTs metabolize the toxic products of lipid peroxidation, damaged DNA and other molecules. Previously, a full-length cDNA of a novel zeta GST gene, PpGST, was characterized from fruit of Pyrus pyrifolia Nakai cv Huobali. In the present study, a constitutive plant expression vector of PpGSTwas constructed and transferred into tobacco (Nicotiana tabacum L. cv Xanthi) to verify the function of PpGST. As a result, the PpGSTgene was successfully integrated into the genome of the transgenic tobacco lines and expressed as expected in the transformants through Southern blotting and quantitative reverse transcription-polymerase chain reaction analysis. Growth of T1 generation plants of PpGST transgenic lines and WT under non-stressful conditions was similar, however, the transgenic tobacco lines showed relatively normal growth under drought, NaCl, and cadmium (Cd) stresses. Furthermore, the T1 transgenic tobacco lines showed significantly slower superoxide anion production rate than the WT under abiotic stress. Simultaneously, the MDA content of each T1 transgenic tobacco plant was only slightly increased and significantly lower than that of the WT under drought, salt and Cd stress. Together with the GST activity of the transgenic tobacco lines, which was significantly increased under stressful conditions, as compared with that in WT, overexpression of PpGSTin tobacco enhanced the tolerance of transgenic tobacco lines to oxidative damage caused by drought, NaCl, and Cd stresses. PMID:24466748
Liu, D; Liu, Y; Rao, J; Wang, G; Li, H; Ge, F; Chen, C
Multiple strategies for gene transfer, expression, knockdown, and chromatin influence in mammalian cell lines and transgenic animals
Manipulation of the eukaryotic genome has contributed to the progress in our knowledge of multicellular organisms but has\\u000a also ameliorated our experimental strategies. Biological questions can now be addressed with more efficiency and reproducibility.\\u000a There are new and varied strategies for gene transfer and sequence manipulation with improved methodologies that facilitate\\u000a the acquisition of results. Cellular systems and transgenic animals
Fķlix Recillas-Targa
Breeding of Selectable Marker-Free Transgenic Rice Lines Containing AP1 Gene with Enhanced Disease Resistance
In order to obtain marker-free transgenic rice with improved disease resistance, the AP1 gene of Capsicum annuum and hygromycin-resistance gene (HPT) were cloned into the two separate T-DNA regions of the binary vector pSB130, respectively, and introduced into the calli derived from the immature seeds of two elite japonica rice varieties, Guangling Xiangjing and Wuxiangjing 9, mediated by Agrobacterium-mediated transformation.
Heng-xiu YU; Qiao-quan LIU; Ling WANG; Zhi-peng ZHAO; Li XU; Ben-li HUANG; Zhi-yun GONG; Shu-zhu TANG; Ming-hong GU
Ablation of cellular prion protein does not ameliorate abnormal neural network activity or cognitive dysfunction in the J20 line of human amyloid precursor protein transgenic mice.
Previous studies suggested that the cellular prion protein (PrP(c)) plays a critical role in the pathogenesis of Alzheimer's disease (AD). Specifically, amyloid-? (A?) oligomers were proposed to cause synaptic and cognitive dysfunction by binding to PrP(c). To test this hypothesis, we crossed human amyloid precursor protein (hAPP) transgenic mice from line J20 onto a PrP(c)-deficient background. Ablation of PrP(c) did not prevent the premature mortality and abnormal neural network activity typically seen in hAPPJ20 mice. Furthermore, hAPPJ20 mice with or without PrP(c) expression showed comparably robust abnormalities in learning and memory and in other behavioral domains at 6-8 months of age. Notably, these abnormalities are not refractory to therapeutic manipulations in general: they can be effectively prevented by interventions that prevent A?-dependent neuronal dysfunction also in other lines of hAPP transgenic mice. Thus, at least in this model, PrP(c) is not an important mediator of A?-induced neurological impairments. PMID:21775587
Cissķ, Moustapha; Sanchez, Pascal E; Kim, Daniel H; Ho, Kaitlyn; Yu, Gui-Qiu; Mucke, Lennart
Comparative Analysis of Osteogenic/Chondrogenic Differentiation Potential in Primary Limb Bud-Derived and C3H10T1/2 Cell Line-Based Mouse Micromass Cultures
Murine micromass models have been extensively applied to study chondrogenesis and osteogenesis to elucidate pathways of endochondral bone formation. Here we provide a detailed comparative analysis of the differentiation potential of micromass cultures established from either BMP-2 overexpressing C3H10T1/2 cells or mouse embryonic limb bud-derived chondroprogenitor cells, using micromass cultures from untransfected C3H10T1/2 cells as controls. Although the BMP-2 overexpressing C3H10T1/2 cells failed to form chondrogenic nodules, cells of both models expressed mRNA transcripts for major cartilage-specific marker genes including Sox9, Acan, Col2a1, Snorc, and Hapln1 at similar temporal sequence, while notable lubricin expression was only detected in primary cultures. Furthermore, mRNA transcripts for markers of osteogenic differentiation including Runx2, Osterix, alkaline phosphatase, osteopontin and osteocalcin were detected in both models, along with matrix calcification. Although the adipogenic lineage-specific marker gene FABP4 was also expressed in micromass cultures, Oil Red O-positive cells along with PPAR?2 transcripts were only detected in C3H10T1/2-derived micromass cultures. Apart from lineage-specific marker genes, pluripotency factors (Nanog and Sox2) were also expressed in these models, reflecting on the presence of various mesenchymal lineages as well as undifferentiated cells. This cellular heterogeneity has to be taken into consideration for the interpretation of data obtained by using these models.
Takacs, Roland; Matta, Csaba; Somogyi, Csilla; Juhasz, Tamas; Zakany, Roza
Regulatory region of the vitellogenin receptor gene sufficient for high-level, germ line cell-specific ovarian expression in transgenic Aedes aegypti mosquitoes.
Vitellogenin receptor (VgR) is responsible for the receptor-mediated endocytosis of vitellogenin (Vg) in the egg formation of an oviparous animal, including insects. Little is known about regulation of VgR gene expression. We analyzed the upstream region of the VgR gene from Aedes aegypti (AaVgR) to identify regulatory elements responsible for its expression in germ cell-specific ovarian expression. Experiments with genetic transformation using the transgene containing the 1.5-Kb upstream portion of the AaVgR gene fused with DsRed and the piggyBac vector showed that this regulatory region is sufficient for correct female and ovary-specific expression of the transgene. This 1.5-Kb upstream region contained binding sites for the ecdysone regulatory hierarchy early gene products E74 and BR-C, as well as transcription factors determining correct tissue- and stage-specific expression of GATA and HNF3/fkh. In situ hybridization demonstrated that in the ovaries of transgenic females DsRed mRNA was present in ovarian germ cells and nurse cells of mature ovarian follicles, together with VgR mRNA. In contrast, DsRed mRNA was absent in the oocyte that had a high level of endogenous VgR mRNA. Although the 1.5-Kb upstream region was sufficient to drive a high-level germ line cell-specific expression of the reporter, additional signals were required for translocation of exogenous mRNA from nurse cells into the oocyte. PMID:16551541
Cho, Kook-Ho; Cheon, Hyang-Mi; Kokoza, Vladimir; Raikhel, Alexander S
Comparative Analysis of Targeted Differentiation of Human Induced Pluripotent Stem Cells (hiPSCs) and Human Embryonic Stem Cells Reveals Variability Associated With Incomplete Transgene Silencing in Retrovirally Derived hiPSC Lines
Functional hepatocytes, cardiomyocytes, neurons, and retinal pigment epithelial (RPE) cells derived from human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) could provide a defined and renewable source of human cells relevant for cell replacement therapies, drug discovery, toxicology testing, and disease modeling. In this study, we investigated the differences between the differentiation potentials of three hESC lines, four retrovirally derived hiPSC lines, and one hiPSC line derived with the nonintegrating Sendai virus technology. Four independent protocols were used for hepatocyte, cardiomyocyte, neuronal, and RPE cell differentiation. Overall, cells differentiated from hESCs and hiPSCs showed functional similarities and similar expression of genes characteristic of specific cell types, and differences between individual cell lines were also detected. Reactivation of transgenic OCT4 was detected specifically during RPE differentiation in the retrovirally derived lines, which may have affected the outcome of differentiation with these hiPSCs. One of the hiPSC lines was inferior in all directions, and it failed to produce hepatocytes. Exogenous KLF4 was incompletely silenced in this cell line. No transgene expression was detected in the Sendai virus-derived hiPSC line. These findings highlight the problems related to transgene expression in retrovirally derived hiPSC lines.
Toivonen, Sanna; Ojala, Marisa; Hyysalo, Anu; Ilmarinen, Tanja; Rajala, Kristiina; Pekkanen-Mattila, Mari; Aanismaa, Riikka; Lundin, Karolina; Palgi, Jaan; Weltner, Jere; Trokovic, Ras; Silvennoinen, Olli; Skottman, Heli; Narkilahti, Susanna; Aalto-Setala, Katriina
An indicator gene for detection of germline retrotransposition in transgenic Drosophila demonstrates RNA-mediated transposition of the LINE I element.
We have marked a cloned Drosophila transposable element--the I element--with an engineered neomycin-containing indicator gene, whose expression is conditioned by passage of the transposon through an RNA intermediate. Mobility of the marked element introduced into Drosophila as a transgene could be detected in vivo, upon in toto selection of developing embryos on G418-containing medium. For resistant individuals, Southern blot analysis and nucleotide sequencing after PCR amplification disclosed transposition of the marked element into new loci, with target site duplications and splicing out of the intron in the indicator gene. It demonstrates that the I element, which is closely related to the widespread mammalian LINEs, transposes through an RNA intermediate, as up to now only conjectured from sequence singularities of this class of 'non-viral retrotransposons'. The developed indicator gene provides a potent new genetic tool for detection and quantitative analysis of retrotransposon mobility and its regulation as it occurs in vivo. Images
Jensen, S; Heidmann, T
Agrobacterium-mediated transformation of safflower and the efficient recovery of transgenic plants via grafting
Background Safflower (Carthamus tinctorius L.) is a difficult crop to genetically transform being susceptible to hyperhydration and poor in vitro root formation. In addition to traditional uses safflower has recently emerged as a broadacre platform for the production of transgenic products including modified oils and pharmaceutically active proteins. Despite commercial activities based on the genetic modification of safflower, there is no method available in the public domain describing the transformation of safflower that generates transformed T1 progeny. Results An efficient and reproducible protocol has been developed with a transformation efficiency of 4.8% and 3.1% for S-317 (high oleic acid content) and WT (high linoleic acid content) genotypes respectively. An improved safflower transformation T-DNA vector was developed, including a secreted GFP to allow non-destructive assessment of transgenic shoots. Hyperhydration and necrosis of Agrobacterium-infected cotyledons was effectively controlled by using iota-carrageenan, L-cysteine and ascorbic acid. To overcome poor in vitro root formation for the first time a grafting method was developed for safflower in which ~50% of transgenic shoots develop into mature plants bearing viable transgenic T1 seed. The integration and expression of secreted GFP and hygromycin genes were confirmed by PCR, Southern and Western blot analysis. Southern blot analysis in nine independent lines indicated that 1-7 transgenes were inserted per line and T1 progeny displayed Mendelian inheritance. Conclusions This protocol demonstrates significant improvements in both the efficiency and ease of use over existing safflower transformation protocols. This is the first complete method of genetic transformation of safflower that generates stably-transformed plants and progeny, allowing this crop to benefit from modern molecular applications.
Analysis of T-DNA integration and generative segregation in transgenic winter triticale (x Triticosecale Wittmack)
Background While the genetic transformation of the major cereal crops has become relatively routine, to date only a few reports were published on transgenic triticale, and robust data on T-DNA integration and segregation have not been available in this species. Results Here, we present a comprehensive analysis of stable transgenic winter triticale cv. Bogo carrying the selectable marker gene HYGROMYCIN PHOSPHOTRANSFERASE (HPT) and a synthetic green fluorescent protein gene (gfp). Progeny of four independent transgenic plants were comprehensively investigated with regard to the number of integrated T-DNA copies, the number of plant genomic integration loci, the integrity and functionality of individual T-DNA copies, as well as the segregation of transgenes in T1 and T2 generations, which also enabled us to identify homozygous transgenic lines. The truncation of some integrated T-DNAs at their left end along with the occurrence of independent segregation of multiple T-DNAs unintendedly resulted in a single-copy segregant that is selectable marker-free and homozygous for the gfp gene. The heritable expression of gfp driven by the maize UBI-1 promoter was demonstrated by confocal laser scanning microscopy. Conclusions The used transformation method is a valuable tool for the genetic engineering of triticale. Here we show that comprehensive molecular analyses are required for the correct interpretation of phenotypic data collected from the transgenic plants.
Development of a convenient in vivo hepatotoxin assay using a transgenic zebrafish line with liver-specific DsRed expression.
Previously we have developed a transgenic zebrafish line (LiPan) with liver-specific red fluorescent protein (DsRed) expression under the fabp10a promoter. Since red fluorescence in the liver greatly facilitates the observation of liver in live LiPan fry, we envision that the LiPan zebrafish may provide a useful tool in analyses of hepatotoxicity based on changes of liver red fluorescence intensity and size. In this study, we first tested four well-established hepatotoxins (acetaminophen, aspirin, isoniazid and phenylbutazone) in LiPan fry and demonstrated that these hepatotoxins could significantly reduce both liver red fluorescence and liver size in a dosage-dependent manner, thus the two measurable parameters could be used as indicators of hepatotoxicity. We then tested the LiPan fry with nine other chemicals including environmental toxicants and human drugs. Three (mefenamic acid, lindane, and arsenate) behave like hepatotoxins in reduction of liver red fluorescence, while three others (17?-estradiol, TCDD [2,3,7,8-tetrachlorodibenzo-p-dioxin] and NDMA [N-nitrosodimethylamine]) caused increase of liver red fluorescence and the liver size. Ethanol and two other chemicals, amoxicillin (antibiotics) and chlorphenamine (pain killer) did not resulted in significant changes of liver red fluorescence and liver size. By quantitative RT-PCR analysis, we found that the changes of red fluorescence intensity caused by different chemicals correlated to the changes of endogenous fabp10a RNA expression, indicating that the measured hepatotoxicity was related to fatty acid transportation and metabolism. Finally we tested a mixture of four hepatotoxins and observed a significant reduction of red fluorescence in the liver at concentrations below the lowest effective concentrations of individual hepatotoxins, suggesting that the transgenic zebrafish assay is capable of reporting compound hepatotoxicity effect from chemical mixtures. Thus, the LiPan transgenic fry provide a rapid and convenient in vivo hepatotoxicity assay that should be applicable to high-throughput hepatotoxicity test in drug screening as well as in biomonitoring environmental toxicants. PMID:24626481
Zhang, Xiaoyan; Li, Caixia; Gong, Zhiyuan
TCreERT2, a Transgenic Mouse Line for Temporal Control of Cre-Mediated Recombination in Lineages Emerging from the Primitive Streak or Tail Bud
The study of axis extension and somitogenesis has been greatly advanced through the use of genetic tools such as the TCre mouse line. In this line, Cre is controlled by a fragment of the T (Brachyury) promoter that is active in progenitor cells that reside within the primitive streak and tail bud and which give rise to lineages emerging from these tissues as the embryonic axis extends. However, because TCre-mediated recombination occurs early in development, gene inactivation can result in an axis truncation that precludes the study of gene function in later or more posterior tissues. To address this limitation, we have generated an inducible TCre transgenic mouse line, called TCreERT2, that provides temporal control, through tamoxifen administration, in all cells emerging from the primitive streak or tail bud throughout development. TCreERT2 activity is mostly silent in the absence of tamoxifen and, in its presence, results in near complete recombination of emerging mesoderm from E7.5 through E13.5. We demonstrate the utility of the TCreERT2 line for determining rate of posterior axis extension and somite formation, thus providing the first in vivo tool for such measurements. To test the usefulness of TCreERT2 for genetic manipulation, we demonstrate that an early deletion of ▀-Catenin via TCreERT2 induction phenocopies the TCre-mediated deletion of ▀-Catenin defect, whereas a later induction bypasses this early phenotype and produces a similar defect in more caudal tissues. TCreERT2 provides a useful and novel tool for the control of gene expression of emerging embryonic lineages throughout development.
Anderson, Matthew J.; Naiche, L. A.; Wilson, Catherine P.; Elder, Cindy; Swing, Deborah A.; Lewandoski, Mark
Cell Suspension Culture-Mediated Incorporation of the Rice Bel Gene into Transgenic Cotton
Cotton plants engineered for resistance to the herbicides, glyphosate or glufosinate have made a considerable impact on the production of the crop worldwide. In this work, embryogenic cell cultures derived from Gossypium hirsutum L. cv Coker 312 hypocotyl callus were transformed via Agrobacterium tumefaciens with the rice cytochrome P450 gene, CYP81A6 (bel). In rice, bel has been shown to confer resistance to both bentazon and sulfanylurea herbicides. Transformed cells were selected on a liquid medium supplemented alternately or simultaneously with kanamycin (50mg/L) and bentazon (4.2 Ąmol). A total of 17 transgenic cotton lines were recovered, based on the initial resistance to bentazon and on PCR detection of the bel transgene. Bel integration into the cotton genome was confirmed by Southern blot and expression of the transgene was verified by RT-PCR. In greenhouse and experimental plot trials, herbicide (bentazon) tolerance of up to 1250mg/L was demonstrated in the transgenic plants. Transgenic lines with a single copy of the bel gene showed normal Mendelian inheritance of the characteristic. Importantly, resistance to bentazon was shown to be stably incorporated in the T1, T2 and T3 generations of self-fertilised descendents and in plants outcrossed to another upland cotton cultivar. Engineering resistance to bentazon in cotton through the heterologous expression of bel opens the possibility of incorporating this trait into elite cultivars, a strategy that would give growers a more flexible alternative to weed management in cotton crops.
Yu, Xiushuang; Sun, Jie; Jones, Brian; Pan, Gang; Cheng, Xiaofei; Wang, Huizhong; Zhu, Shuijin; Sun, Yuqiang
Cell suspension culture-mediated incorporation of the rice bel gene into transgenic cotton.
Cotton plants engineered for resistance to the herbicides, glyphosate or glufosinate have made a considerable impact on the production of the crop worldwide. In this work, embryogenic cell cultures derived from Gossypium hirsutum L. cv Coker 312 hypocotyl callus were transformed via Agrobacterium tumefaciens with the rice cytochrome P450 gene, CYP81A6 (bel). In rice, bel has been shown to confer resistance to both bentazon and sulfanylurea herbicides. Transformed cells were selected on a liquid medium supplemented alternately or simultaneously with kanamycin (50mg/L) and bentazon (4.2 Ąmol). A total of 17 transgenic cotton lines were recovered, based on the initial resistance to bentazon and on PCR detection of the bel transgene. Bel integration into the cotton genome was confirmed by Southern blot and expression of the transgene was verified by RT-PCR. In greenhouse and experimental plot trials, herbicide (bentazon) tolerance of up to 1250 mg/L was demonstrated in the transgenic plants. Transgenic lines with a single copy of the bel gene showed normal Mendelian inheritance of the characteristic. Importantly, resistance to bentazon was shown to be stably incorporated in the T1, T2 and T3 generations of self-fertilised descendents and in plants outcrossed to another upland cotton cultivar. Engineering resistance to bentazon in cotton through the heterologous expression of bel opens the possibility of incorporating this trait into elite cultivars, a strategy that would give growers a more flexible alternative to weed management in cotton crops. PMID:22768325
Ke, Liping; Liu, RuiE; Chu, Bijue; Yu, Xiushuang; Sun, Jie; Jones, Brian; Pan, Gang; Cheng, Xiaofei; Wang, Huizhong; Zhu, Shuijin; Sun, Yuqiang
Development of oral epithelial cell line ROE2 with differentiation potential from transgenic rats harboring temperature-sensitive simian virus40 large T-antigen gene.
We have developed an immortalized oral epithelial cell line, ROE2, from fetal transgenic rats harboring temperature-sensitive simian virus 40 large T-antigen gene. The cells grew continuously at either a permissive temperature of 33░C or an intermediate temperature of 37░C. At the nonpermissive temperature of 39░C, on the other hand, growth decreased significantly, and the Sub-G1 phase of the cell cycle increased, indicating that the cells undergo apoptosis at a nonpermissive temperature. Histological and immunocytochemical analyses revealed that ROE2 cells at 37░C had a stratified epithelial-like morphology and expressed cytokeratins Krt4 and Krt13, marker proteins for oral nonkeratinized epithelial cells. Global-scale comprehensive microarray analysis, coupled with bioinformatics tools, demonstrated a significant gene network that was obtained from the upregulated genes. The gene network contained 16 genes, including Cdkn1a, Fos, Krt13, and Prdm1, and was associated mainly with the biological process of skin development in the category of biological functions, organ development. These four genes were validated by quantitative real-time polymerase chain reaction, and the results were nearly consistent with the microarray data. It is therefore anticipated that this cell line will be useful as an in vitro model for studies such as physiological functions, as well as for gene expression in oral epithelial cells. PMID:24521861
Tabuchi, Yoshiaki; Wada, Shigehito; Ikegame, Mika; Kariya, Ayako; Furusawa, Yukihiro; Hoshi, Nobuhiko; Yunoki, Tatsuya; Suzuki, Nobuo; Takasaki, Ichiro; Kondo, Takashi; Suzuki, Yoshihisa
The substantive equivalence of transgenic (Bt and Chi) and non-transgenic cotton based on metabolite profiles.
Compositional studies comparing transgenic with non-transgenic counterpart plants are almost universally required by governmental regulatory bodies. In the present study, two T(2) transgenic cotton lines containing chitinase (Line 11/57) and Bt lines (Line 61) were compared with non-transgenic counterpart. To do this, biochemical characteristics of leaves and seeds, including amino acids, fatty acids, carbohydrates, anions, and cations contents of the studied lines were analyzed using GC/MS, high-performance liquid chromatography (HPLC), and ion chromatography (IC) analyzers, respectively. polymerase chain reaction (PCR) and Western blot analyses confirmed the presence and expression of Chi and Bt genes in the studied transgenic lines. Although, compositional analysis of leaves contents confirmed no significant differences between transgenic and non-transgenic counterpart lines, but it was shown that glucose content of chitinase lines, fructose content of transgenic lines (Bt and chitinase) and asparagine and glutamine of chitinase lines were significantly higher than the non-transgenic counterpart plants. Both the transgenic lines (Bt and chitinase) showed significant decrease in the amounts of sodium in comparison to the non-transgenic counterpart plants. The experiments on the seeds showed that histidine, isoleucine, leucine, and phenylalanine contents of all transgenic and non-transgenic lines were the same, whereas other amino acids were significantly increased in the transgenic lines. Surprisingly, it was observed that the concentrations of stearic acid, myristic acid, oleic acid, and linoleic acid in the chitinase line were significantly different than those of non-transgenic counterpart plants, but these components were the same in both Bt line and its non-transgenic counterpart. It seems that more changes observed in the seed contents than leaves is via this point that seeds are known as metabolites storage organs, so they show greater changes in the metabolites contents comparing to the leaves. PMID:24374853
Modirroosta, Bentol Hoda; Tohidfar, Masoud; Saba, Jalal; Moradi, Foad
Comparison of transgenic Gerbera hybrida lines and traditional varieties shows no differences in cytotoxicity or metabolic fingerprints
Genetic modification using gene transfer (GM) is still controversial when applied to plant breeding at least in Europe. One\\u000a major concern is how GM affects other genes and thus the metabolism of the plant. In this study, 225 genetically modified\\u000a lines of the ornamental plant Gerbera hybrida and 42 non-GM gerbera varieties were used to investigate changes in secondary metabolism.
Miia Marika Ainasoja; Leena Lyydia Pohjala; Põivi Sirpa Marjaana Tammela; Panu Juhani Somervuo; Pia Maarit Vuorela; Teemu Heikki Teeri
Transgenic mice expressing a partially deleted gene for type I procollagen (COL1A1). A breeding line with a phenotype of spontaneous fractures and decreased bone collagen and mineral.
A line of transgenic mice was prepared that expressed moderate levels of an internally deleted human gene for the pro alpha 1(I) chain of type I procollagen. The gene construct was modeled after a sporadic in-frame deletion of the human gene that produced a lethal variant of osteogenesis imperfecta by causing biosynthesis of shortened pro alpha 1(I) chains. 89 transgenic mice from the line were examined. About 6% had a lethal phenotype with extensive fractures at birth, and 33% had fractures but were viable. The remaining 61% of the transgenic mice had no apparent fractures as assessed by x ray examination on the day of birth. Brother-sister matings produced eight litters in which approximately 40% of the mice had the lethal phenotype, an observation indicating that expression of the exogenous gene was more lethal in putative homozygous mice from the line. Examination of femurs from the transgenic mice indicated that the bones were significantly shorter in length and had a decrease in wet weight, mineral content, and collagen content. However, there was no statistically significant change in the mineral to collagen ratio. Biomechanical measurements on femurs from the mice at 6 wk indicated a decrease in force and energy to failure. There was also a decrease in strain to failure and an increase in Young's modulus of elasticity, observations indicating increased brittleness of bone matrix. The results suggested that the transgenic mice may be an appropriate model for testing potential therapies for osteogenesis imperfecta. They may also be a useful model for studying osteoporosis. Images
Pereira, R; Khillan, J S; Helminen, H J; Hume, E L; Prockop, D J
Large-scale production of enhancer trapping lines for rice functional genomics
Using a rapid and efficient Agrobacterium tumefaciens mediated rice transformation protocol, we had got about 100,000 independent enhancer trapping lines of japonica rice variety Nipponbare. T1 seeds had already been obtained from 60,000 lines. Regeneration of transgenic plants could be as short as 3 months starting from callus induction, thus the occurrence of somaclonal variation was reduced to a minimum
Yongzhi Yang; Hao Peng; Hongmei Huang; Jinxia Wu; Shirong Jia; Dafang Huang; Tiegang Lu
Institute for Systems Biology: T1DBase
NSDL National Science Digital Library
T1DBase was created jointly by the Institute for Systems Biology, the Juvenile Diabetes Research Foundation International, and the Juvenile Diabetes Research Foundation/Wellcome Trust Diabetes and Inflammation Laboratory to support the work of Type 1 diabetes (T1D) researchers. T1DBase currently "includes annotated genomic sequences for suspected T1D susceptibility regions; microarray data; functional annotation of genes active in beta cells; and 'global' datasets, generally from the literature, that are useful for systems biology studies." Information is available for human, rat, and mouse Type 1 diabetes candidate regions. The site also offers several tools including Gbrowse, Cytoscape, Beta Cell Gene Expression Bank, and Kegg Pathways. T1DBase was created as a community resource, and the website invites researchers to contribute both ideas and data.
T1 mapping in ischaemic heart disease.
A unique feature of cardiac magnetic resonance is its ability to characterize myocardium. Proton relaxation times, T1, T2, and T2* are a reflection of the composition of individual tissues, and change in the presence of disease. Research into T1 mapping has largely been focused in the study of cardiomyopathies, but T1 mapping also shows huge potential in the study of ischaemic heart disease. In fact, the first cardiac T1 maps were used to characterize myocardial infarction. Robust high-resolution myocardial T1 mapping is now available for use as a clinical tool. This quantitative technique is simple to perform and analyse, minimally subjective, and highly reproducible. This review aims to summarize the present state of research on the topic, and to show the clinical potential of this method to aid the diagnosis and treatment of patients with ischaemic heart disease. PMID:24566951
h-Ici, Darach O; Jeuthe, Sarah; Al-Wakeel, Nadya; Berger, Felix; Kuehne, Titus; Kozerke, Sebastian; Messroghli, Daniel R
A DNA insertional mutation results in microphthalmia in transgenic mice
Transgenic mice were produced by microinjection of a humanA?-globin gene construct containing site 2 of the locus control region and theA?-globin gene with its 3? enhancer sequence. One transgenic mouse line 95?HS2?en91) displayed an altered phenotype when the insertion event of this transgenic line was homozygous. These animals lack the normal pigmentation seen in their hemizygous and non-transgenic littermates, thus
Joan M. Krakowsky; Raymond E. Boissy; Jon C. Neumann; Jerry B. Lingrel
Transcription-dependent silencing of inducible convergent transgenes in transgenic mice
Background Silencing of transgenes in mice is a common phenomenon typically associated with short multi-copy transgenes. We have investigated the regulation of the highly inducible human granulocyte-macrophage colony-stimulating-factor gene (Csf2) in transgenic mice. Results In the absence of any previous history of transcriptional activation, this transgene was expressed in T lineage cells at the correct inducible level in all lines of mice tested. In contrast, the transgene was silenced in a specific subset of lines in T cells that had encountered a previous episode of activation. Transgene silencing appeared to be both transcription-dependent and mediated by epigenetic mechanisms. Silencing was accompanied by loss of DNase I hypersensitive sites and inability to recruit RNA polymerase II upon stimulation. This pattern of silencing was reflected by increased methylation and decreased acetylation of histone H3 K9 in the transgene. We found that silenced lines were specifically associated with a single pair of tail-to-tail inverted repeated copies of the transgene embedded within a multi-copy array. Conclusions Our study suggests that epigenetic transgene silencing can result from convergent transcription of inverted repeats which can lead to silencing of an entire multi-copy transgene array. This mechanism may account for a significant proportion of the reported cases of transgene inactivation in mice.
Immortalization of epididymal epithelium in transgenic mice expressing simian virus 40 T antigen: characterization of cell lines and regulation of the polyoma enhancer activator 3.
In the present study epididymal epithelium was immortalized in transgenic mice by expressing simian virus 40 T antigen under a 5.0-kb mouse glutathione peroxidase 5 promoter (GPX5-Tag1). Epididymal tumorigenesis was associated with an increase in c-Myc expression, and a marked decrease in B-Myc expression, with a 500-fold lower level in the GPX5-Tag1 caput epididymis compared with wild-type caput. Furthermore, B-Myc was undetectable in the immortalized corpus and cauda epididymis. Hence, it is possible that the normally high B-Myc expression in the epididymis is one of the factors contributing to the highly resistant nature of epididymis toward immortalization. Morphologically different epithelial cell lines were generated from the immortalized epididymides, and the cells expressed several genes typical for epididymal epithelium, such as mouse epididymal 1, mouse epididymal protein 9, androgen and estrogen receptors, anion exchangers 2 and 4, retinoic acid receptor alpha, and polyoma enhancer activator 3 (PEA3). This indicated the differentiated status of the cells and their usefulness for analyzing epididymal gene expression in vitro. As PEA3 is considered to be one of the transcription factors responsible for epididymal gene expression, we further studied its regulation in epididymal cells in vitro. The data showed that PEA3 mRNA expression is regulated in the epididymis via protein kinase A and ERK signaling cascades. Inhibiting protein kinase A resulted in up-regulation and inhibiting ERK resulted in down-regulation of PEA3 mRNA, whereas no significant effect on PEA3 expression was found by modulating the protein kinase C, stress-activated p38, phosphoinositol 3-kinase and p70 S6 kinase cascades. PMID:14527890
Sipilõ, Petra; Shariatmadari, Ramin; Huhtaniemi, Ilpo T; Poutanen, Matti
Expression of Cry1Ab Protein in a Marker-Free Transgenic Bt Rice Line and Its Efficacy in Controlling a Target Pest, Chilo suppressalis (Lepidoptera: Crambidae).
A marker-free Bt transgenic rice line, mfb-MH86, was recently developed in China, which contains a cry1Ab gene driven by a ubiquitin promoter. This Bt gene confers resistance to a range of lepidopteran species, including the striped stem borer, Chilo suppressalis (Walker). The expression of Cry1Ab protein in mfb-MH86 leaves, stems and leaf sheaths (hereinafter referred to as stems), and roots was evaluated throughout the rice-growing season using an enzyme-linked immunosorbent assay. In addition, mfb-MH86 resistance to C. suppressalis, a major pest of rice, was evaluated in a laboratory bioassay with field-collected rice stems. Cry1Ab protein levels of mfb-MH86 were highest in leaves (9.71-34.09 ?g/g dry weight [DW]), intermediate in stems (7.66-18.51 ?g/g DW), and lowest in roots (1.95-13.40 ?g/g DW). In all tissues, Cry1Ab levels in mfb-MH86 were higher in seedling and tillering stages than in subsequent growth stages. In the laboratory bioassay, mortality of C. suppressalis after 6 d of feeding on mfb-MH86 stems was 100% throughout the rice-growing season; mortality of C. suppressalis when feeding on stems of the nontransformed isoline, MH86, ranged from 15.0 to 38.3%. The results indicate that Cry1Ab protein levels in mfb-MH86 stems are sufficient to protect plants against C. suppressalis throughout the rice-growing season. Although our results are promising, further comprehensive evaluations of mfb-MH86, including field surveys, will be needed before commercial use. PMID:24495566
Wang, Yanan; Zhang, Lei; Li, Yunhe; Liu, Yanmin; Han, Lanzhi; Zhu, Zhen; Wang, Feng; Peng, Yufa
Improved Nutritive Quality and Salt Resistance in Transgenic Maize by Simultaneously Overexpression of a Natural Lysine-Rich Protein Gene, SBgLR, and an ERF Transcription Factor Gene, TSRF1.
Maize (Zea mays L.), as one of the most important crops in the world, is deficient in lysine and tryptophan. Environmental conditions greatly impact plant growth, development and productivity. In this study, we used particle bombardment mediated co-transformation to obtain marker-free transgenic maize inbred X178 lines harboring a lysine-rich protein gene SBgLR from potato and an ethylene responsive factor (ERF) transcription factor gene, TSRF1, from tomato. Both of the target genes were successfully expressed and showed various expression levels in different transgenic lines. Analysis showed that the protein and lysine content in T1 transgenic maize seeds increased significantly. Compared to non-transformed maize, the protein and lysine content increased by 7.7% to 24.38% and 8.70% to 30.43%, respectively. Moreover, transgenic maize exhibited more tolerance to salt stress. When treated with 200 mM NaCl for 48 h, both non-transformed and transgenic plant leaves displayed wilting and losing green symptoms and dramatic increase of the free proline contents. However, the degree of control seedlings was much more serious than that of transgenic lines and much more increases of the free proline contents in the transgenic lines than that in the control seedlings were observed. Meanwhile, lower extent decreases of the chlorophyll contents were detected in the transgenic seedlings. Quantitative RT-PCR was performed to analyze the expression of ten stress-related genes, including stress responsive transcription factor genes, ZmMYB59 and ZmMYC1, proline synthesis related genes, ZmP5CS1 and ZmP5CS2, photosynthesis-related genes, ZmELIP, ZmPSI-N, ZmOEE, Zmrbcs and ZmPLAS, and one ABA biosynthesis related gene, ZmSDR. The results showed that with the exception of ZmP5CS1 and ZmP5CS2 in line 9-10 and 19-11, ZmMYC1 in line 19-11 and ZmSDR in line 19-11, the expression of other stress-related genes were inhibited in transgenic lines under normal conditions. After salt treatment, the expressions of the ten stress-related genes were significantly induced in both wild-type (WT) and transgenic lines. However, compared to WT, the increases of ZmP5CS1 in all these three transgenic lines and ZmP5CS2 in line 9-10 were less than WT plants. This study provides an effective approach of maize genetic engineering for improved nutritive quality and salt tolerance. PMID:23629675
Wang, Meizhen; Liu, Chen; Li, Shixue; Zhu, Dengyun; Zhao, Qian; Yu, Jingjuan
A model for the mechanism of precise integration of a microinjected transgene
A unique transgenic mouse line has undergone transgene integration in a very precise fashion. The phenotype displayed by mice of the line followed the predicted inheritance patterns for X-linked transgene insertion which has been confirmed. In order to investigate the mechanism of integration the DNA sequence of the transgene and cellular junctions have been determined. A comparison between wild type
Morag McFarlane; Joanna B. Wilson
Transgenic mice in developmental toxicology
Advances in molecular biology and embryology are being utilized for the generation of transgenic mice, animals that contain specific additions, deletions, or modifications of genes or sequences in their DNA. Mouse embryonic stem cells and homologous recombination procedures have made it possible to target specific DNA structural alterations to highly localized region in the host chromosomes. The majority of the DNA structural rearrangements in transgenic mice can be passed through the germ line and used to establish new genetic traits in the carrier animals. Since the use of transgenic mice is having such an enormous impact on so many areas of mammalian biological research, including developmental toxicology, the objective of this review is to briefly describe the fundamental methodologies for generating transgenic mice and to describe one particular application that has direct relevance to the field of genetic toxicology.
Woychik, R.P.
Transgenic mice in developmental toxicology
Advances in molecular biology and embryology are being utilized for the generation of transgenic mice, animals that contain specific additions, deletions, or modifications of genes or sequences in their DNA. Mouse embryonic stem cells and homologous recombination procedures have made it possible to target specific DNA structural alterations to highly localized region in the host chromosomes. The majority of the DNA structural rearrangements in transgenic mice can be passed through the germ line and used to establish new genetic traits in the carrier animals. Since the use of transgenic mice is having such an enormous impact on so many areas of mammalian biological research, including developmental toxicology, the objective of this review is to briefly describe the fundamental methodologies for generating transgenic mice and to describe one particular application that has direct relevance to the field of genetic toxicology.
Woychik, R.P.
SirT1 in muscle physiology and disease: lessons from mouse models
Sirtuin 1 (SirT1) is the largest of the seven members of the sirtuin family of class III nicotinamide adenine dinucleotide (NAD+)-dependent protein deacetylases, whose activation is beneficial for metabolic, neurodegenerative, inflammatory and neoplastic diseases, and augments life span in model organisms (Finkel et al., 2009; Lavu et al., 2008). In vitro studies show that SirT1 protects genome integrity and is involved in circadian physiological rhythms (Asher et al., 2008; Nakahata et al., 2008; Oberdoerffer et al., 2008). In the last few years, a fundamental role for SirT1 in the metabolism and differentiation of skeletal muscle cells has been uncovered (Fulco et al., 2003), and the use of specific transgenic or knockout SirT1 mouse models implicates it in the protection of heart muscle from oxidative and hypertrophic stresses (Alcendor et al., 2007). In this Perspective, we review the recent exciting findings that have established a key role for the ÆlongevityÆ protein SirT1 in skeletal and heart muscle physiology and disease. Furthermore, given the multiple biological functions of SirT1, we discuss the unique opportunities that SirT1 mouse models can offer to improve our integrated understanding of the metabolism, as well as the regeneration and aging-associated changes in the circadian function, of skeletal and heart muscle.
Vinciguerra, Manlio; Fulco, Marcella; Ladurner, Andreas; Sartorelli, Vittorio; Rosenthal, Nadia
Every silver lining has a cloud: the scientific and animal welfare issues surrounding a new approach to the production of transgenic animals.
The scientific basis and advantages of using recently developed CRISPR/Cas-9 technology for transgenesis have been assessed with respect to other production methods, laboratory animal welfare, and the scientific relevance of transgenic models of human diseases in general. As the new technology is straightforward, causes targeted DNA double strand breaks and can result in homozygous changes in a single step, it is more accurate and more efficient than other production methods and speeds up transgenesis. CRISPR/Cas-9 also obviates the use of embryonic stem cells, and is being used to generate transgenic non-human primates (NHPs). While the use of this method reduces the level of animal wastage resulting from the production of each new strain, any long-term contribution to reduction will be offset by the overall increase in the numbers of transgenic animals likely to result from its widespread usage. Likewise, the contribution to refinement of using a more-precise technique, thereby minimising the occurrence of unwanted genetic effects, will be countered by a probable substantial increase in the production of transgenic strains of increasingly sentient species. For ethical and welfare reasons, we believe that the generation of transgenic NHPs should be allowed only in extremely exceptional circumstances. In addition, we present information, which, on both welfare and scientific grounds, leads us to question the current policy of generating ever-more new transgenic models in light of the general failure of many of them, after over two decades of ubiquitous use, to result in significant advances in the understanding and treatment of many key human diseases. Because this unsatisfactory situation is likely to be due to inherent, as well as possibly avoidable, limitations in the transgenic approach to studying disease, which are briefly reviewed, it is concluded that a thorough reappraisal of the rationale for using genetically-altered animals in fundamental research and by the pharmaceutical industry, and for its support by funding bodies, should be undertaken. In the meantime, the use of CRISPR/Cas-9 to generate new transgenic cells in culture is to be guardedly encouraged. PMID:24901907
Combes, Robert D; Balls, Michael
Brain segmentation performance using T1-weighted images versus T1 maps
NASA Astrophysics Data System (ADS)
The recent driven equilibrium single-pulse observation of T1 (DESPOT1) approach permits real-time clinical acquisition of large-volume and high-isotropic-resolution T1 mapping of MR tissue parameters with improved uniformity. It is assumed that the quantitative nature of maps will facilitate clinical applications such as disease diagnosis and comparison across subjects. However, there is not yet enough quantitative evidence on the actual benefit of adopting T1 maps, especially in computer-aided medical image analysis tasks. In this study, we compare methods with respect to image types, T1-weighted images or T1 maps, in automatic brain MRI segmentation. Our experimental results demonstrate that, using T1 maps, different segmentation algorithms show better agreement with each other, compared to that from using T1-weighted images. Furthermore, through multi-dimensional-scaling projection, we are able to visualize the relative affinity among segmentation results, which reveals that the projections of those segmentations using two different types of input images tend to form two separate clusters. Finally, by comparing to expert segmented reference segmentation of brain sub-regions, our results clearly indicate a better agreement between the manual reference and those automatic ones on T1 maps. In other words, our study provides an evidence for the hypothesis that compared to the conventionally used T1-weighted images, T1 maps lead to improved reliability in automatic brain MRI segmentation task.
Li, Xiaoxing; Wyatt, Christopher
Thermodynamics of ribonuclease T1 denaturation.
Differential scanning calorimetry has been used to investigate the thermodynamics of denaturation of ribonuclease T1 as a function of pH over the pH range 2-10, and as a function of NaCl and MgCl2 concentration. At pH 7 in 30 mM PIPES buffer, the thermodynamic parameters are as follows: melting temperature, T1/2 = 48.9 +/- 0.1 degrees C; enthalpy change, delta H = 95.5 +/- 0.9 kcal mol-1; heat capacity change, delta Cp = 1.59 kcal mol-1 K-1; free energy change at 25 degrees C, delta G degrees (25 degrees C) = 5.6 kcal mol-1. Both T1/2 = 56.5 degrees C and delta H = 106.1 kcal mol-1 are maximal near pH 5. The conformational stability of ribonuclease T1 is increased by 3.0 kcal/mol in the presence of 0.6 M NaCl or 0.3 M MgCl2. This stabilization results mainly from the preferential binding of cations to the folded conformation of the protein. The estimates of the conformational stability of ribonuclease T1 from differential scanning calorimetry are shown to be in remarkably good agreement with estimates derived from an analysis of urea denaturation curves. PMID:1591247
Hu, C Q; Sturtevant, J M; Thomson, J A; Erickson, R E; Pace, C N
T1 Genes Which Affect Transduction
Amber mutants of T1 were grown on each of three donor strains which were identical except that they carried different suppressors: respectively, supD, supE, and supB. The efficiency with which the mutants were able to transduce was tested after growth on each donor. In general, it was found that functions which control the synthesis of phage DNA usually caused significant increases in the efficiency of transduction (EOT). A few mutants located in genes essential for head production caused significant decreases in EOT. The presence of a particular suppressor in a donor can cause noteworthy changes in the EOT by certain of the mutant phages. Amber mutations in gene 3 of T1 were extremely sensitive to the particular suppressor present in the donor, showing a 17-fold decrease in EOT compared with other mutants after growth in donors with the supD suppressor and a 75-fold increase after growth in supE donors. Increases in EOT by early genes of T1 do not seem to be caused by a lack of competition of bacterial DNA with phage DNA during packaging since, in most instances, infective phage were produced in relatively normal amounts compared with wild-type T1. Phage DNA synthesis and degradations of the host chromosome are closely coupled in T1 infections; we believe that increases in EOT by mutants of early functions are due to inefficient degradation of the host chromosome. Images
Borchert, L. D.; Drexler, H.
Epigenetic mechanisms affect mutant p53 transgene expression in WAP-mutp53 transgenic mice.
We describe the construction and phenotypic characterization of 23 whey acidic protein (WAP)-mutp53 transgenic mouse lines. The mutp53-expressing lines showed a mosaic expression pattern for the transgenes, leading to a heterogeneous yet mouse line-specific expression pattern for mutp53 upon induction. Only few lines were obtained, in which the majority of the induced mammary epithelial cells expressed the mutp53 transgene, most of the transgenic lines did not express mutp53, or expressed the transgene in less than 2% of the induced mammary epithelial cells. Hormone requirements for mutp53 transgene expression from the WAP-promoter differed in high and low expressing lines, being low in high expressing lines, and even lower in multiparous mutp53 mice, where persistent expression of the transgene occurred. Repeated induction of mutp53 expression through repeated parturition resulted in the formation of expanding mutp53-expressing foci within the mammary alveolar epithelium. The data suggest that epigenetic mechanisms play a role in modulating the expression of the mutp53 transgene. To support this idea, we crossed a nonexpressing WAP-mutp53 line with a strongly SV40 T-antigen-expressing WAP-T mouse line. In the bitransgenic mice, T-antigen-induced chromatin remodeling led to re-expression of epigenetically silenced mutp53 transgene(s). In these mice, mutp53 expression was much more variable compared to SV40 T-antigen expression, and seemed to depend on the coexpression of SV40 T-antigen. Mutp53 expression in this system thus resembles the situation in many human tumors, where one can observe a heterogeneous expression of mutp53, despite a homogeneous distribution of the p53 mutation in the tumor cells. PMID:15870706
Krepulat, Frauke; L÷hler, J³rgen; Heinlein, Christina; Hermannstõdter, Andrea; Tolstonog, Genrich V; Deppert, Wolfgang
Pre-selection of integration sites imparts repeatable transgene expression
Variable gene expression amongst transgenic lines occurs due to copy number and to random associa- tions of incoming DNA with chromosomal elements at the site of integration. Here we describe a method of identifying sites permissive for transgene expression and their use for efficient introduction of single copy transgenes by homologous recombination. ES clones were selected in HAT medium for
Helen Wallace; Ray Ansell; John Clark; Jim McWhir
Stability of transgene expression as a challenge for genetic engineering
The major genetic factors affecting the expression of newly introduced genes in transgenic plants including epigenetic effects are summarized. Examples of (trans)gene silencing and the genetic signals involved are given. Based on current knowledge, several strategies to generate stable transgenic lines can be followed. Although initial laboratory and field tests over few generations allow good predictions about the long-term expression
Antje Dietz-Pfeilstetter
Spatial and temporal expression of the Cre gene under the control of the MMTV-LTR in different lines of transgenic mice
Cre-loxP based gene deletion approaches hold great promise to enhance our understanding of molecular pathways controlling mammary development and breast cancer. We reported earlier the generation of transgenic mice that express the Cre recombinase under the control of the mouse mammary tumor virus (MMTV) long terminal repeat (LTR). These mice have become a valuable research tool to delete genes specifically
Kay-Uwe Wagner; Toni Ward; Barbara Davis; Roger Wiseman; Lothar Hennighausen
A transgenic mouse line with a 58-kb fragment deletion in chromosome 11E1 that encompasses part of the Fam20a gene and its upstream region shows growth disorder.
Growth disorder is an umbrella term for a range of abnormal growth patterns, such as unusually fast or slow growth in infants or children. The causes of growth disorder include hormonal irregularities, chronic disease, complications during pregnancy or genetic conditions. A complex trait such as body size is influenced by multiple genes as well as environmental factors, giving rise to a continuous spectrum of phenotypes. This causal complexity makes discovery of the genetic determinants of growth disorder rather difficult. We here report our discovery of a transgenic mouse line exhibiting growth disorder, which we happened to discover in the course of generating transgenic mice expressing a viral gene. Although these mice did not express any corresponding viral mRNA or protein due to a deletion in the transgene, they showed slow growth in the 5 weeks after birth and ceased growing thereafter, while maintaining a weight equivalent to that of 3-week-old normal mice. Histopathological analysis of the organs of these mice revealed that malnutrition and metabolic disorder occurred at 5 weeks after birth in the liver. Genetic analysis has revealed that the growth disorder is associated with a 58-kb fragment deletion in chromosome 11E1 that encompasses part of the Fam20a gene and part of its upstream region. The present study thus points out for the first time the possible link between Fam20a mutation and growth disorder. PMID:20847595
An, Chunying; Ide, Yoshihiro; Nagano-Fujii, Motoko; Kitazawa, Sohei; Shoji, Ikuo; Hotta, Hak
A field-grown transgenic tomato line expressing higher levels of polyamines reveals legume cover crop mulch-specific perturbations in fruit phenotype at the levels of metabolite profiles, gene expression, and agronomic characteristics
Genetic modification of crop plants to introduce desirable traits such as nutritional enhancement, disease and pest resistance, and enhanced crop productivity is increasingly seen as a promising technology for sustainable agriculture and boosting food production in the world. Independently, cultural practices that utilize alternative agriculture strategies including organic cultivation subscribe to sustainable agriculture by limiting chemical usage and reduced tillage. How the two together affect fruit metabolism or plant growth in the field or whether they are compatible has not yet been tested. Fruit-specific yeast S-adenosylmethionine decarboxylase (ySAMdc) line 579HO, and a control line 556AZ were grown in leguminous hairy vetch (Vicia villosa Roth) (HV) mulch and conventional black polyethylene (BP) mulch, and their fruit analysed. Significant genotypeūmulch-dependent interactions on fruit phenotype were exemplified by differential profiles of 20 fruit metabolites such as amino acids, sugars, and organic acids. Expression patterns of the ySAMdc transgene, and tomato SAMdc, E8, PEPC, and ICDHc genes were compared between the two lines as a function of growth on either BP or HV mulch. HV mulch significantly stimulated the accumulation of asparagine, glutamate, glutamine, choline, and citrate concomitant with a decrease in glucose in the 556AZ fruits during ripening as compared to BP. It enables a metabolic system in tomato somewhat akin to the one in higher polyamine-accumulating transgenic fruit that have higher phytonutrient content. Finally, synergism was found between HV mulch and transgenic tomato in up-regulating N:C indicator genes PEPC and ICDHc in the fruit.
Neelam, Anil; Cassol, Tatiana; Mehta, Roshni A.; Abdul-Baki, Aref A.; Sobolev, Anatoli P.; Goyal, Ravinder K.; Abbott, Judith; Segre, Anna L.; Handa, Avtar K.; Mattoo, Autar K.
High expression of transgene protein in Spirodela.
The monocot family Lemnaceae (duckweed) is composed of small, edible, aquatic plants. Spirodela oligorrhiza SP is a duckweed with a biomass doubling time of about 2 days under controlled, axenic conditions. Stably transformed Spirodela plants were obtained following co-cultivation of regenerative calli with Agrobacterium tumefaciens. GFP activity was successfully monitored in different subcellular compartments of the plant and correlated with different targeting sequences. Transgenic lines were followed for a period of at least 18 months and more than 180 vegetative doublings (generations). The lines are stable in morphology, growth rate, transgene expression, and activity as measured by DNA-DNA and immunoblot hybridizations, fluorescence activity measurements, and antibiotic resistance. The level of transgene expression is a function of leader sequences rather than transgene copy number. A stable, transgenic, GFP expression level >25% of total soluble protein is demonstrated for the S. oligorrhiza system, making it among the higher expressing systems for nuclear transformation in a higher plant. PMID:17492286
Vunsh, Ron; Li, Jihong; Hanania, Uri; Edelman, Marvin; Flaishman, Moshe; Perl, Avihai; Wisniewski, Jean-Pierre; Freyssinet, Georges
Overexpression of the rat sarcoplasmic reticulum Ca2+ ATPase gene in the heart of transgenic mice accelerates calcium transients and cardiac relaxation.
The Ca2+ ATPase of the sarcoplasmic reticulum (SERCA2) plays a dominant role in lowering cytoplasmic calcium levels during cardiac relaxation and reduction of its activity has been linked to delayed diastolic relaxation in hypothyroid and failing hearts. To determine the contractile alterations resulting from increased SERCA2 expression, we generated transgenic mice overexpressing a rat SERCA2 transgene. Characterization of a heterozygous transgenic mouse line (CJ5) showed that the amount of SERCA2 mRNA and protein increased 2. 6-fold and 1.2-fold, respectively, relative to control mice. Determination of the relative synthesis rate of SERCA2 protein showed an 82% increase. The mRNA levels of some of the other genes involved in calcium handling, such as the ryanodine receptor and calsequestrin, remained unchanged, but the mRNA levels of phospholamban and Na+/Ca2+ exchanger increased 1.4-fold and 1.8-fold, respectively. The increase in phospholamban or Na+/Ca2+ exchanger mRNAs did not, however, result in changes in protein levels. Functional analysis of calcium handling and contractile parameters in isolated cardiac myocytes indicated that the intracellular calcium decline (t1/2) and myocyte relengthening (t1/2) were accelerated by 23 and 22%, respectively. In addition, the rate of myocyte shortening was also significantly faster. In isolated papillary muscle from SERCA2 transgenic mice, the time to half maximum postrest potentiation was significantly shorter than in negative littermates. Furthermore, cardiac function measured in vivo, demonstrated significantly accelerated contraction and relaxation in SERCA2 transgenic mice that were further augmented in both groups with isoproterenol administration. Similar results were obtained for the contractile performance of myocytes isolated from a separate line (CJ2) of homozygous SERCA2 transgenic mice. Our findings suggest, for the first time, that increased SERCA2 expression is feasible in vivo and results in enhanced calcium transients, myocardial contractility, and relaxation that may have further therapeutic implications.
He, H; Giordano, F J; Hilal-Dandan, R; Choi, D J; Rockman, H A; McDonough, P M; Bluhm, W F; Meyer, M; Sayen, M R; Swanson, E; Dillmann, W H
Immortalization of Epididymal Epithelium in Transgenic Mice Expressing Simian Virus 40 T Antigen: Characterization of Cell Lines and Regulation of the Polyoma Enhancer Activator 3
In the present study epididymal epithelium was immortalized in transgenic mice by expressing simian virus 40 T antigen under a 5.0-kb mouse glutathione peroxidase 5 promoter (GPX5-Tag1). Epididymal tumorigenesis was associated with an increase in c-Myc expression, and a marked decrease in B-Myc expression, with a 500-fold lower level in the GPX5- Tag1 caput epididymis compared with wild-type caput. Fur-
Efficient production of germline transgenic chickens using lentiviral vectors
An effective method for genetic modification of chickens has yet to be developed. An efficient technology, enabling production of transgenic birds at high frequency and with reliable expression of transgenes, will have many applications, both in basic research and in biotechnology. We investigated the efficiency with which lentiviral vectors could transduce the chicken germ line and examined the expression of introduced reporter transgenes. Ten founder cockerels transmitted the vector to between 4% and 45% of their offspring and stable transmission to the G2 generation was demonstrated. Analysis of expression of reporter gene constructs in several transgenic lines showed a conserved expression profile between individuals that was maintained after transmission through the germ line. These data demonstrate that lentiviral vectors can be used to generate transgenic lines with an efficiency in the order of 100-fold higher than any previously published method, with no detectable silencing of transgene expression between generations.
McGrew, Michael J; Sherman, Adrian; Ellard, Fiona M; Lillico, Simon G; Gilhooley, Hazel J; Kingsman, Alan J; Mitrophanous, Kyriacos A; Sang, Helen
Distinct Human and Mouse Membrane Trafficking Systems for Sweet Taste Receptors T1r2 and T1r3
The sweet taste receptors T1r2 and T1r3 are included in the T1r taste receptor family that belongs to class C of the G protein-coupled receptors. Heterodimerization of T1r2 and T1r3 is required for the perception of sweet substances, but little is known about the mechanisms underlying this heterodimerization, including membrane trafficking. We developed tagged mouse T1r2 and T1r3, and human T1R2 and T1R3 and evaluated membrane trafficking in human embryonic kidney 293 (HEK293) cells. We found that human T1R3 surface expression was only observed when human T1R3 was coexpressed with human T1R2, whereas mouse T1r3 was expressed without mouse T1r2 expression. A domain-swapped chimera and truncated human T1R3 mutant showed that the Venus flytrap module and cysteine-rich domain (CRD) of human T1R3 contain a region related to the inhibition of human T1R3 membrane trafficking and coordinated regulation of human T1R3 membrane trafficking. We also found that the Venus flytrap module of both human T1R2 and T1R3 are needed for membrane trafficking, suggesting that the coexpression of human T1R2 and T1R3 is required for this event. These results suggest that the Venus flytrap module and CRD receive taste substances and play roles in membrane trafficking of human T1R2 and T1R3. These features are different from those of mouse receptors, indicating that human T1R2 and T1R3 are likely to have a novel membrane trafficking system.
Shimizu, Madoka; Goto, Masao; Kawai, Takayuki; Yamashita, Atsuko; Kusakabe, Yuko
The distribution of transgene insertion sites in barley determined by physical and genetic mapping.
The exact site of transgene insertion into a plant host genome is one feature of the genetic transformation process that cannot, at present, be controlled and is often poorly understood. The site of transgene insertion may have implications for transgene stability and for potential unintended effects of the transgene on plant metabolism. To increase our understanding of transgene insertion sites in barley, a detailed analysis of transgene integration in independently derived transgenic barley lines was carried out. Fluorescence in situ hybridization (FISH) was used to physically map 23 transgene integration sites from 19 independent barley lines. Genetic mapping further confirmed the location of the transgenes in 11 of these lines. Transgene integration sites were present only on five of the seven barley chromosomes. The pattern of transgene integration appeared to be nonrandom and there was evidence of clustering of independent transgene insertion events within the barley genome. In addition, barley genomic regions flanking the transgene insertion site were isolated for seven independent lines. The data from the transgene flanking regions indicated that transgene insertions were preferentially located in gene-rich areas of the genome. These results are discussed in relation to the structure of the barley genome.
Salvo-Garrido, Haroldo; Travella, Silvia; Bilham, Lorelei J; Harwood, Wendy A; Snape, John W
{open_quotes}Horizontal{close_quotes} gene transfer from a transgenic potato line to a bacterial pathogen (Erwinia chrysanthemi) occurs - if at all - at an extremely low frequency
The frequency of possible {open_quotes}horizontal{close_quotes} gene transfer between a plant and a tightly associated bacterial pathogen was studied in a model system consisting of transgenic Solanum tuberosum, containing a {beta}-lactamase gene linked to a pBR322 origin of replication, and Erwinia chrysanthemi. This experimental system offers optimal conditions for the detection of possible horizontal gene transfer events, even when they occur at very low frequency. Horizontal gene transfer was not detected under conditions mimicking a {open_quotes}natural{close_quotes} infection. The gradual, stepwise alteration of artificial, positive control conditions to idealized natural conditions, however, allowed the characterization of factors that affected gene transfer, and revealed a gradual decrease of the gene transfer frequency from 6.3 x 10{sup -2} under optimal control conditions to a calculated 2.0 x 10{sub -17} under idealized natural conditions. These data, in combination with other published studies, argue that horizontal gene transfer is so rare as to be essentially irrelevant to any realistic assessment of the risk involved in release experiments involving transgenic plants. 22 refs., 3 figs., 2 tabs.
Schlueter, K.; Fuetterer, J.; Potrykus, I. [Institute of Plant Sciences, Zuerich (Switzerland)] [Institute of Plant Sciences, Zuerich (Switzerland)
Expression of the Shpx2 peroxidase gene of Stylosanthes humilis in transgenic tobacco leads to enhanced resistance to Phytophthora parasitica pv. nicotianae and Cercospora nicotianae.
Abstract Previous research indicated that the constitutive expression of a pathogen-inducible peroxidase gene (Shpx6a) from Stylosanthes humilis in transgenic plants resulted in enhanced resistance to fungal pathogens ( Kazan, K., Goulter, K.C., Way, H.M. and Manners, J.M. (1998) Expression of a pathogenesis-related peroxidase of Stylosanthes humilis in transgenic tobacco and canola and its effect on disease development. Plant Sci. 136, 207-217). We have now investigated another pathogen-inducible peroxidase gene of S. humilis, termed Shpx2, which is highly divergent from Shpx6a. Constitutive expression of the Shpx2 cDNA was obtained in tobacco using the 35S CaMV promoter, and up to a 12-fold increase in total peroxidase activity was observed in the leaves of transgenic plants compared to nontransgenic controls. Disease development was evaluated after inoculations in T(1) and T(2) transgenic lines expressing Shpx2. Lesion expansion was significantly (P < 0.05) reduced by up to 25% and 50% on leaves and stems, respectively, of transgenic plants expressing high levels of peroxidase compared to nontransgenic controls, following inoculation with Phytophthora parasitica pv. nicotianae, the cause of black shank disease. In addition, plant survival and recovery were greatly enhanced in transgenic plants following stem inoculations of plants with this plant pathogen. A significant (55%, P < 0.05) reduction in lesion number was observed in transgenic plants with high levels of peroxidase activity following inoculation with the fungus Cercospora nicotianae, the cause of frog-eye disease. No significant differences in disease development were observed between the lines expressing Shpx2 and controls following inoculation with the bacterium Pseudomonas syringae pv. tabaci, the cause of wildfire disease. These results provide evidence for a role for this peroxidase gene in plant defence, and suggest that diverse peroxidase genes may be employed as components of strategies aimed at the engineering of disease resistance. PMID:20572969
Way, H M; Kazan, K; Goulter, K C; Birch, R G; Manners, J M
[Progress in transgenic fish techniques and application].
Transgenic technique provides a new way for fish breeding. Stable lines of growth hormone gene transfer carps, salmon and tilapia, as well as fluorescence protein gene transfer zebra fish and white cloud mountain minnow have been produced. The fast growth characteristic of GH gene transgenic fish will be of great importance to promote aquaculture production and economic efficiency. This paper summarized the progress in transgenic fish research and ecological assessments. Microinjection is still the most common used method, but often resulted in multi-site and multi-copies integration. Co-injection of transposon or meganuclease will greatly improve the efficiency of gene transfer and integration. "All fish" gene or "auto gene" should be considered to produce transgenic fish in order to eliminate misgiving on food safety and to benefit expression of the transferred gene. Environmental risk is the biggest obstacle for transgenic fish to be commercially applied. Data indicates that transgenic fish have inferior fitness compared with the traditional domestic fish. However, be-cause of the genotype-by-environment effects, it is difficult to extrapolate simple phenotypes to the complex ecological interactions that occur in nature based on the ecological consequences of the transgenic fish determined in the laboratory. It is critical to establish highly naturalized environments for acquiring reliable data that can be used to evaluate the environ-mental risk. Efficacious physical and biological containment strategies remain to be crucial approaches to ensure the safe application of transgenic fish technology. PMID:21586396
Ye, Xing; Tian, Yuan-Yuan; Gao, Feng-Ying
Controlling transgene expression in subcutaneous implants using a skin lotion containing the apple metabolite phloretin
Adjustable control of therapeutic transgenes in engineered cell implants after transdermal and topical delivery of nontoxic trigger molecules would increase convenience, patient compliance, and elimination of hepatic first-pass effect in future therapies. Pseudomonas putida DOT-T1E has evolved the flavonoid-triggered TtgR operon, which controls expression of a multisubstrate-specific efflux pump (TtgABC) to resist plant-derived defense metabolites in its rhizosphere habitat. Taking advantage of the TtgR operon, we have engineered a hybrid P. putida¢mammalian genetic unit responsive to phloretin. This flavonoid is contained in apples, and, as such, or as dietary supplement, regularly consumed by humans. The engineered mammalian phloretin-adjustable control element (PEACE) enabled adjustable and reversible transgene expression in different mammalian cell lines and primary cells. Due to the short half-life of phloretin in culture, PEACE could also be used to program expression of difficult-to-produce protein therapeutics during standard bioreactor operation. When formulated in skin lotions and applied to the skin of mice harboring transgenic cell implants, phloretin was able to fine-tune target genes and adjust heterologous protein levels in the bloodstream of treated mice. PEACE-controlled target gene expression could foster advances in biopharmaceutical manufacturing as well as gene- and cell-based therapies.
Gitzinger, Marc; Kemmer, Christian; El-Baba, Marie Daoud; Weber, Wilfried; Fussenegger, Martin
Controlling transgene expression in subcutaneous implants using a skin lotion containing the apple metabolite phloretin.
Adjustable control of therapeutic transgenes in engineered cell implants after transdermal and topical delivery of nontoxic trigger molecules would increase convenience, patient compliance, and elimination of hepatic first-pass effect in future therapies. Pseudomonas putida DOT-T1E has evolved the flavonoid-triggered TtgR operon, which controls expression of a multisubstrate-specific efflux pump (TtgABC) to resist plant-derived defense metabolites in its rhizosphere habitat. Taking advantage of the TtgR operon, we have engineered a hybrid P. putida-mammalian genetic unit responsive to phloretin. This flavonoid is contained in apples, and, as such, or as dietary supplement, regularly consumed by humans. The engineered mammalian phloretin-adjustable control element (PEACE) enabled adjustable and reversible transgene expression in different mammalian cell lines and primary cells. Due to the short half-life of phloretin in culture, PEACE could also be used to program expression of difficult-to-produce protein therapeutics during standard bioreactor operation. When formulated in skin lotions and applied to the skin of mice harboring transgenic cell implants, phloretin was able to fine-tune target genes and adjust heterologous protein levels in the bloodstream of treated mice. PEACE-controlled target gene expression could foster advances in biopharmaceutical manufacturing as well as gene- and cell-based therapies. PMID:19549857
Gitzinger, Marc; Kemmer, Christian; El-Baba, Marie Daoud; Weber, Wilfried; Fussenegger, Martin
Generation and Characterization of an Nse-CreERT2 Transgenic Line Suitable for Inducible Gene Manipulation in Cerebellar Granule Cells
We created an Nse-CreERT2 mouse line expressing the tamoxifen-inducible CreERT2 recombinase under the control of the neuron-specific enolase (Nse) promoter. By using Cre reporter lines we could show that this Nse-CreERT2 line has recombination activity in the granule cells of all cerebellar lobules as well as in postmitotic granule cell precursors in the external granular layer of the developing cerebellum. A few hippocampal dentate gyrus granule cells showed Cre-mediated recombination as well. Cre activity could be induced in both the developing and adult mouse brain. The established mouse line constitutes a valuable tool to study the function of genes expressed by cerebellar granule cells in the developing and adult brain. In combination with reporter lines it is a useful model to analyze the development and maintenance of the cerebellar architecture including granule cell distribution, migration, and the extension of granule cell fibers in vivo.
Pohlkamp, Theresa; Steller, Laura; May, Petra; Gunther, Thomas; Schule, Roland; Frotscher, Michael
Arabidopsis rd29A::DREB1A enhances freezing tolerance in transgenic potato
The freezing tolerance of 38 independent transgenic potato lines derived from the cultivar Desiree was tested in vitro using\\u000a plantlets. The lines were transgenic for the DREB1A gene under control of the rd29A promoter, both of which were derived from Arabidopsis thaliana. The level of damage caused by freezing varied significantly among the transgenic clones and a non-transgenic control (cv.
Babak Behnam; Akira Kikuchi; Fevziye Celebi-Toprak; Mie Kasuga; Kazuko Yamaguchi-Shinozaki; Kazuo N. Watanabe
Iridium 192 implantation of T1 and T2 carcinomas of the mobile tongue
Between 1970 and 1986, 166 patients with T1 or T2 epidermoid carcinomas of the mobile tongue were treated by iridium 192 implantation (70 T1N0, 83 T2N0, 13 T1-2 N1-3). Five-year actuarial survival was 52% for T1N0, 44% for T2aN0, and 8% for or T1-2 N1-3. Cause specific survivals were 90%, 71%, and 46%, respectively. Local control was 87% for both T1N0 and T2N0, and 69% for T1-2 N1-3. Seven of 23 failures were salvaged by surgery, increasing local control to 96% for T1 and 90% for T2. Thirty-six patients developed a minor or moderate necrosis (16% T1, 28% T2). Half of these involved bone but only five required surgical intervention. Both local control (LC) and necrosis (nec) increased with increasing dose but improvement beyond 65 Gy is minimal (less than or equal to 60 Gy: LC = 78% nec = 13%; 65 Gy: LC = 90% nec = 29%; greater than or equal to 70 Gy: LC = 94% nec = 23%). For N0 patients, neck management consisted of surveillance (n = 78), elective neck dissection followed with external irradiation for pathologically positive nodes (n = 72), or irradiation (n = 3). Clinically positive nodes (13 patients) were managed by either neck dissection followed by external irradiation if pathologically positive (n = 10) or irradiation alone (n = 3). Regional control was 79% for N0 patients, improving to 88% after surgical salvage, and was 9/13 for N1-3 patients. We recommend that T1 and T2 carcinomas of the mobile tongue be treated by iridium 192 implantation to deliver 65 Gy. Mandibular necrosis should be reduced by using an intra-oral lead-lined dental mold.
Mazeron, J.J.; Crook, J.M.; Benck, V.; Marinello, G.; Martin, M.; Raynal, M.; Haddad, E.; Peynegre, R.; Le Bourgeois, J.P.; Walop, W. (Hopital Henri Mondor, Creteil (France))
Silent information regulator (Sir)T1 inhibits NF-?B signaling to maintain normal skeletal remodeling.
Silent information regulator T1 (SirT1) is linked to longevity and negatively controls NF-?B signaling, a crucial mediator of survival and regulator of both osteoclasts and osteoblasts. Here we show that NF-?B repression by SirT1 in both osteoclasts and osteoblasts is necessary for proper bone remodeling and may contribute to the mechanisms linking aging and bone loss. Osteoclast- or osteoblast-specific SirT1 deletion using the Sirt(flox/flox) mice crossed to lysozyme M-cre and the 2.3?kb col1a1-cre transgenic mice, respectively, resulted in decreased bone mass caused by increased resorption and reduced bone formation. In osteoclasts, lack of SirT1 promoted osteoclastogenesis in vitro and activated NF-?B by increasing acetylation of Lysine 310. Importantly, this increase in osteoclastogenesis was blocked by pharmacological inhibition of NF-?B. In osteoblasts, decreased SirT1 reduced osteoblast differentiation, which could also be rescued by inhibition of NF-?B. In further support of the critical role of NF-?B signaling in bone remodeling, elevated NF-?B activity in I?B?(+/-) mice uncoupled bone resorption and formation, leading to reduced bone mass. These findings support the notion that SirT1 is a genetic determinant of bone mass, acting in a cell-autonomous manner in both osteoblasts and osteoclasts, through control of NF-?B and bone cell differentiation. PMID:23172686
Edwards, James R; Perrien, Daniel S; Fleming, Nicole; Nyman, Jeffry S; Ono, Koichiro; Connelly, Linda; Moore, Megan M; Lwin, Seint T; Yull, Fiona E; Mundy, Gregory R; Elefteriou, Florent
Transgene expression in the striatum following intracerebral injections of DNA nanoparticles encoding for human glial cell line-derived neurotrophic factor (hGDNF)
A goal of our studies is to develop a potential therapeutic for ParkinsonÆs disease (PD) by a human GDNF (hGDNF) expression plasmid administered to the rat striatum as a compacted DNA nanoparticle (DNP) and which will generate long-term hGDNF expression at biologically active levels. In the present study we used a DNA plasmid encoding for hGDNF and a polyubiquitin C (UbC) promoter that was previously shown to have activity in both neurons and glia, but primarily in glia. A two-fold improvement was observed at the highest plasmid dose when using hGDNF DNA incorporating sequences found in RNA splice variant 1 compared to splice variant 2; of note, the splice variant 2 sequence is used in most preclinical studies. This optimized expression cassette design includes flanking scaffold matrix attachment elements (S/MARs) as well as a CpG-depleted prokaryotic domain and, where possible, eukaryotic elements. Stable long-term GDNF activity at levels 300¢400% higher than baseline was observed following a single intracerebral injection. In a previous study DNPs plasmids encoding for reporter genes had been successful in generating long-term reporter transgene activity in the striatum (>365 days) and in this study produced sustained GDNF activity at the longest assessed time point (6 months).
Fletcher, Anita M.; Kowalczyk, Tomasz H.; Padegimas, Linas; Cooper, Mark J.; Yurek, David M.
Sampling Submicron T1 Bacteriophage Aerosols
Liquid impingers, filter papers, and fritted bubblers were partial viable collectors of radioactive submicron T1 bacteriophage aerosols at 30, 55, and 85% relative humidity. Sampler differences for viable collection were due to incomplete physical collection (slippage) and killing of phage by the samplers. Dynamic aerosols of a mass median diameter of 0.2 ? were produced with a Dautrebande generator from concentrated aqueous purified phage suspensions containing extracellular soluble radioactive phosphate as a physical tracer. There was considerable destruction of phage by the Dautrebande generator; phage titers of the Dautrebande suspension decreased exponentially, but there was a progressive (linear) increase in tracer titers. Liquid impingers recovered the most viable phage but allowed considerable (30 to 48%) slippage, which varies inversely with the aerosol relative humidity. Filter papers were virtually complete physical collectors of submicron particles but were the most destructive. Fritted bubbler slippage was more than 80%. With all samplers, phage kill was highest at 85% relative humidity and lowest at 55% relative humidity. An electrostatic precipitator was used to collect aerosol samples for particle sizing with an electron microscope. The particle size was slightly larger at 85% relative humidity than at 30 or 55% relative humidity. Images Fig. 1 Fig. 4
Harstad, J. Bruce
Sampling submicron T1 bacteriophage aerosols.
Liquid impingers, filter papers, and fritted bubblers were partial viable collectors of radioactive submicron T1 bacteriophage aerosols at 30, 55, and 85% relative humidity. Sampler differences for viable collection were due to incomplete physical collection (slippage) and killing of phage by the samplers. Dynamic aerosols of a mass median diameter of 0.2 mu were produced with a Dautrebande generator from concentrated aqueous purified phage suspensions containing extracellular soluble radioactive phosphate as a physical tracer. There was considerable destruction of phage by the Dautrebande generator; phage titers of the Dautrebande suspension decreased exponentially, but there was a progressive (linear) increase in tracer titers. Liquid impingers recovered the most viable phage but allowed considerable (30 to 48%) slippage, which varies inversely with the aerosol relative humidity. Filter papers were virtually complete physical collectors of submicron particles but were the most destructive. Fritted bubbler slippage was more than 80%. With all samplers, phage kill was highest at 85% relative humidity and lowest at 55% relative humidity. An electrostatic precipitator was used to collect aerosol samples for particle sizing with an electron microscope. The particle size was slightly larger at 85% relative humidity than at 30 or 55% relative humidity. PMID:5866038
Harstad, J B
T1D in College (Type 1 Diabetes)
... College for young adults with T1D means more freedom along with increased responsibility. Gain insights and information ... with a strategic plan to progressively remove the impact of T1D from people's lives until it is ...
Establishment and characterization of conditionally immortalized endothelial cell lines from the thoracic duct and inferior vena cava of tsA58\\/EGFP double-transgenic rats
The basic biology of blood vascular endothelial cells has been well documented. However, little is known about that of lymphatic endothelial cells, despite their importance under normal and pathological conditions. The lack of a lymphatic endothelial cell line has hampered progress in this field. The objective of this study has been to establish and characterize lymphatic and venous endothelial cell
Mitsuhiro Matsuo; Keiichi Koizumi; Sanae Yamada; Masatoshi Tomi; Ri-ichi Takahashi; Masatsugu Ueda; Tetsuya Terasaki; Masuo Obinata; Ken-ichi Hosoya; Osamu Ohtani; Ikuo Saiki
Neuroprotective Effects of Glial Cell Line-Derived Neurotrophic Factor Mediated by an Adeno-Associated Virus Vector in a Transgenic Animal Model of Amyotrophic Lateral Sclerosis
Amyotrophic lateral sclerosis (ALS) is a relentlessly progressive lethal disease that involves selective annihilation of motoneu- rons. Glial cell line-derived neurotrophic factor (GDNF) is pro- posed to be a promising therapeutic agent for ALS and other motor neuron diseases. Because adeno-associated virus (AAV) has been developed as an attractive gene delivery system with proven safety, we explored the therapeutic efficacy
Li-Jun Wang; Yan-Yan Lu; Shin-ichi Muramatsu; Kunihiko Ikeguchi; Ken-ichi Fujimoto; Takashi Okada; Hiroaki Mizukami; Takashi Matsushita; Yutaka Hanazono; Akihiro Kume
Quantitative real-time PCR assay to detect transgene copy number in cotton (Gossypium hirsutum).
A TaqMan quantitative real-time PCR detection system was developed to examine transgene copy number in cotton. GhUBC1, a gene validated to be present as a single copy per haploid Gossypium hirsutum genome, was used as the endogenous reference to estimate copy number of GFP and selection marker NPTII in 28 T0 plants. This system was found to be more accurate than genomic Southern blot hybridization and could effectively tell homozygotes from heterozygotes in a T1 transgenic cotton population. Therefore it is suitable for efficient and cost effective early screening of transgenic seedlings and identifying transgene homozygotes in segregation populations. PMID:18078801
Yi, Cheng Xin; Zhang, Jun; Chan, Ka Man; Liu, Xiao Kun; Hong, Yan
Single-Copy Transgenic Mice with Chosen-Site Integration
We describe a general way of introducing transgenes into the mouse germ line for comparing different sequences without the complications of variation in copy number and insertion site. The method uses homologous recombination in embryonic stem (ES) cells to generate mice having a single copy of a transgene integrated into a chosen location in the genome. To test the method,
Sarah K. Bronson; Elizabeth G. Plaehn; Kimberly D. Kluckman; John R. Hagaman; Nobuyo Maeda; Oliver Smithies
Metastatic Prostate Cancer in a Transgenic Mouse1
We have previously reported the developmentof a transgenic mouse model for prostate cancer derived from PB-Tag transgenic line 8247, henceforth designated the TRAMP (trausgenic adenocarcinomamouse prostate) model. We now describe the temporal and spatial consequences of transgeneexpressionand reportthe identificationand characterization of metastaticdisease in the TRAMPmodeLTRAMPmice characteristi cally express the T antigen oncoprotein by 8 weeks of age and develop distinct
Jeffrey R. Gingrich; Roberto J. Barrios; Ronald A. Morton; Brendan F. Boyce; Francesco J. DeMayo; Milton J. Finegold; Roxani Angelopoulou; Jeffrey M. Rosen; Norman M. Greenbere
T1? MRI Quantification of Arthroscopically-Confirmed Cartilage Degeneration
9 asymptomatic subjects and 6 patients underwent T1? MRI to determine whether Outerbridge grade 1 or 2 cartilage degeneration observed during arthroscopy could be detected noninvasively. MRI was performed 2¢3 months post-arthroscopy using sagittal T1-weighted and axial and coronal T1? MRI from which spatial T1? relaxation maps were calculated from segmented T1-weighted images. Median T1? relaxation times of patients with arthroscopically documented cartilage degeneration and asymptomatic subjects were significantly different (p < 0.001) and median T1? exceeded asymptomatic articular cartilage median T1? by 2.5 to 9.2 ms. In 8 observations of mild cartilage degeneration at arthroscopy (Outerbridge grades 1 and 2), mean compartment T1? was elevated in 5, but in all observations, large foci of increased T1? were observed. It was determined that T1? could detect some, but not all, Outerbridge grade 1 and 2 cartilage degeneration but that a larger patient population is needed to determine the sensitivity to these changes.
Witschey, Walter RT; Borthakur, Arijitt; Fenty, Matt; Kneeland, J Bruce; Lonner, Jess H; McArdle, Erin L.; Sochor, Matt; Reddy, Ravinder
Hepatic steatosis in transgenic mice overexpressing human histone deacetylase 1
It is generally thought that histone deacetylases (HDACs) play important roles in the transcriptional regulation of genes. However, little information is available concerning the specific functions of individual HDACs in disease states. In this study, two transgenic mice lines were established which harbored the human HDAC1 gene. Overexpressed HDAC1 was detected in the nuclei of transgenic liver cells, and HDAC1 enzymatic activity was significantly higher in the transgenic mice than in control littermates. The HDAC1 transgenic mice exhibited a high incidence of hepatic steatosis and nuclear pleomorphism. Molecular studies showed that HDAC1 may contribute to nuclear pleomorphism through the p53/p21 signaling pathway.
Wang, Ai-Guo [Laboratory of Human Genomics, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 305-806 (Korea, Republic of); Seo, Sang-Beom [Department of Life Science, College of Natural Sciences, Chung-Ang University, Seoul 156-756 (Korea, Republic of); Moon, Hyung-Bae [Department of Pathology, School of Medicine, Institute of Medical Science, Wonkwang University, Iksan, Jeonbuk 570-749 (Korea, Republic of); Shin, Hye-Jun [Laboratory of Human Genomics, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 305-806 (Korea, Republic of); Kim, Dong Hoon [Department of Oral Biochemistry, College of Dentistry, Chonnam National University, Gwangju 500-757 (Korea, Republic of); Kim, Jin-Man [Department of Bioscience and Biotechnology, Institute of Bioscience, Sejong University, 98 Kunja-dong, Kwangjin-gu, Seoul 143-747 (Korea, Republic of); Lee, Tae-Hoon [Department of Pathology, College of Medicine, Chungnam National University, Daejeon 301-131 (Korea, Republic of); Kwon, Ho Jeong [Department of Oral Biochemistry, College of Dentistry, Chonnam National University, Gwangju 500-757 (Korea, Republic of); Yu, Dae-Yeul [Laboratory of Human Genomics, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 305-806 (Korea, Republic of)]. E-mail: email@example.com; Lee, Dong-Seok [Laboratory of Human Genomics, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 305-806 (Korea, Republic of)]. E-mail: email@example.com
Overexpression of TLR7 promotes cell-intrinsic expansion and autoantibody production by transitional T1 B cells
Toll-like receptor (TLR), a ligand for single-stranded RNA, has been implicated in the development of pathogenic anti-RNA autoantibodies both in systemic lupus erythematous (SLE) patients and in murine models of lupus. It is still unclear, however, where and how TLR7-mediated interactions affect the development of autoreactive B cells. We found that overexpression of TLR7 in transgenic mice (TLR7.1Tg) leads to marked alterations of transitional (T1) B cells, associated with their expansion and proliferation within the splenic red pulp (RP). This phenotype was intrinsic to the T1 subset of B cells and occurred independently of type 1 IFN signals. Overexpression of RNase in TLR7.1Tg mice significantly limited the expansion and proliferation of T1 cells, indicating that endogenous RNA complexes are driving their activation. TLR7.1Tg T1 cells were hyper-responsive to anti-IgM and TLR7 ligand stimulation in vitro and produced high concentrations of class-switched IgG2b and IgG2c, including anti-RNA antibodies. Our results demonstrate that initial TLR7 stimulation of B cells occurs at the T1 stage of differentiation in the splenic RP and suggest that dysregulation of TLR7 expression in T1 cells can result in production of autoantibodies.
Chappell, Craig P.; Sun, Xizhang; Kolhatkar, Nikita; Teal, Thomas H.; Wiedeman, Alice E.; Kim, Jinoh; Tanaka, Lena; Buechler, Matthew B.; Hamerman, Jessica A.; Imanishi-Kari, Thereza; Clark, Edward A.; Elkon, Keith B.
Overexpression of TLR7 promotes cell-intrinsic expansion and autoantibody production by transitional T1 B cells.
Toll-like receptor (TLR), a ligand for single-stranded RNA, has been implicated in the development of pathogenic anti-RNA autoantibodies both in systemic lupus erythematous (SLE) patients and in murine models of lupus. It is still unclear, however, where and how TLR7-mediated interactions affect the development of autoreactive B cells. We found that overexpression of TLR7 in transgenic mice (TLR7.1Tg) leads to marked alterations of transitional (T1) B cells, associated with their expansion and proliferation within the splenic red pulp (RP). This phenotype was intrinsic to the T1 subset of B cells and occurred independently of type 1 IFN signals. Overexpression of RNase in TLR7.1Tg mice significantly limited the expansion and proliferation of T1 cells, indicating that endogenous RNA complexes are driving their activation. TLR7.1Tg T1 cells were hyper-responsive to anti-IgM and TLR7 ligand stimulation in vitro and produced high concentrations of class-switched IgG2b and IgG2c, including anti-RNA antibodies. Our results demonstrate that initial TLR7 stimulation of B cells occurs at the T1 stage of differentiation in the splenic RP and suggest that dysregulation of TLR7 expression in T1 cells can result in production of autoantibodies. PMID:24145511
Giltiay, Natalia V; Chappell, Craig P; Sun, Xizhang; Kolhatkar, Nikita; Teal, Thomas H; Wiedeman, Alice E; Kim, Jinoh; Tanaka, Lena; Buechler, Matthew B; Hamerman, Jessica A; Imanishi-Kari, Thereza; Clark, Edward A; Elkon, Keith B
Cloning of transgenic tobacco BY2 cells; an efficient method to analyse and reduce high natural heterogeneity of transgene expression
BACKGROUND: Phenotypic characterization of transgenic cell lines, frequently used in plant biology studies, is complicated because transgene expression in individual cells is often heterogeneous and unstable. To identify the sources and to reduce this heterogeneity, we transformed tobacco (Nicotiana tabacum L.) BY-2 cells with a gene encoding green fluorescent protein (GFP) using Agrobacterium tumefaciens, and then introduced a simple cloning
Eva Nocarova; Lukas Fischer
Transgene behaviour across two generations in a large random population of transgenic rice plants produced by particle bombardment
The relationship between transgene copy number, rearrangement levels, inheritance patterns, expression levels, transgene stability and plant fertility was analysed in a random population of 95 independently transformed rice plant lines. This analysis has been conducted for both the selectable marker gene (aphIV) and the unselected reporter gene (gusA), in the presence or absence of flanking Matrix Attachment Regions (MARs) in
P. Vain; V. A. James; B. Worland; J. W. Snape
A high-throughput method for quantifying transgene expression in transformed plants with real-time PCR analysis
When analysing plant transformation in plant transgenic lines, determining the level of transgene expression is essential.\\u000a Northern blot analysis and reverse-transcription polymerase chain reaction (RT-PCR) are currently used for this purpose and\\u000a enable qualitative and semiquantitative estimation of transgene mRNA levels. We have introduced a real-time PCR method for\\u000a quantitative determination of transgene expression level in transgenic potato plants containing
N. Toplak; V. OkrÜlar; D. Stani?-Racman; K. Gruden; J. Äel
Competitive Performance of Transgenic Wheat Resistant to Powdery Mildew
Genetically modified (GM) plants offer an ideal model system to study the influence of single genes that confer constitutive resistance to pathogens on the ecological behaviour of plants. We used phytometers to study competitive interactions between GM lines of spring wheat Triticum aestivum carrying such genes and control lines. We hypothesized that competitive performance of GM lines would be reduced due to enhanced transgene expression under pathogen levels typically encountered in the field. The transgenes pm3b from wheat (resistance against powdery mildew Blumeria graminis) or chitinase and glucanase genes from barley (resistance against fungi in general) were introduced with the ubiquitin promoter from maize (pm3b and chitinase genes) or the actin promoter from rice (glucanase gene). Phytometers of 15 transgenic and non-transgenic wheat lines were transplanted as seedlings into plots sown with the same 15 lines as competitive environments and subject to two soil nutrient levels. Pm3b lines had reduced mildew incidence compared with control lines. Chitinase and chitinase/glucanase lines showed the same high resistance to mildew as their control in low-nutrient treatment and slightly lower mildew rates than the control in high-nutrient environment. Pm3b lines were weaker competitors than control lines. This resulted in reduced yield and seed number. The Pm3b line with the highest transgene expression had 53.2% lower yield than the control whereas the Pm3b line which segregated in resistance and had higher mildew rates showed only minor costs under competition. The line expressing both chitinase and glucanase genes also showed reduced yield and seed number under competition compared with its control. Our results suggest that single transgenes conferring constitutive resistance to pathogens can have ecological costs and can weaken plant competitiveness even in the presence of the pathogen. The magnitude of these costs appears related to the degree of expression of the transgenes.
Kalinina, Olena; Zeller, Simon L.; Schmid, Bernhard
L-Theanine elicits umami taste via the T1R1Ā+ĀT1R3 umami taste receptor.
L-Theanine is a unique amino acid present in green tea. It elicits umami taste and has a considerable effect on tea taste and quality. We investigated L-theanine activity on the T1R1Ā+ĀT1R3 umami taste receptor. L-Theanine activated T1R1Ā+ĀT1R3-expressing cells and showed a synergistic response with inosine 5'-monophosphate. The site-directed mutagenesis analysis revealed that L-theanine binds to L-amino acid binding site in the Venus flytrap domain of T1R1. This study shows that L-theanine elicits an umami taste via T1R1Ā+ĀT1R3. PMID:24633359
Narukawa, Masataka; Toda, Yasuka; Nakagita, Tomoya; Hayashi, Yukako; Misaka, Takumi
Enhanced salt tolerance of transgenic progeny of tall fescue (Festuca arundinacea) expressing a vacuolar Na+/H+ antiporter gene from Arabidopsis.
Salinity is a major abiotic stress factor limiting crop production. To generate salt-tolerant turf and forage, we had transformed tall fescue (Festuca arundinacea) with AtNHX1, a vacuolar Na(+)/H(+) antiporter gene from Arabidopsis thaliana. In this paper, we report that overexpression of the AtNHX1 gene confers enhanced salt tolerance to the transformed tall fescue progenies. DNA gel blot analysis and reverse transcription (RT) polymerase chain reaction (PCR) were carried out to confirm the inheritance and expression of the AtNHX1 gene in transgenic T(1) and T(2) lines. These transgenic lines showed no phenotypic changes or yield reduction. Plants carrying the AtNHX1 gene were more resistant to a 20 mM NaCl solution than control plants. The roots of the transgenic lines had a higher sodium content than controls, due to an increased Na(+)/H(+) antiporter activity in tonoplast vesicles. Our results suggest that this accumulation of sodium in vacuoles of root cells, mediated by vacuolar Na(+)/H(+) antiporters, reduced the toxic effects of salinity to tall fescue and thus enhanced its salt tolerance. PMID:17561307
Zhao, Junsheng; Zhi, Daying; Xue, Zheyong; Liu, Heng; Xia, Guangmin
Marker-free transgenic (MFT) near-isogenic introgression lines (NIILs) of 'golden' indica rice (cv. IR64) with accumulation of provitamin A in the endosperm tissue.
We have developed near-isogenic introgression lines (NIILs) of an elite indica rice cultivar (IR64) with the genes for beta-carotene biosynthesis from dihaploid (DH) derivatives of golden japonica rice (cv. T309). A careful analysis of the DH lines indicated the integration of the genes of interest [phytoene synthase (psy) and phytoene desaturase (crtI)] and the selectable marker gene (hygromycin phosphotransferase, hph) in two unlinked loci. During subsequent crossing, progenies could be obtained carrying only the locus with psy and crtI, which was segregated independently from the locus containing the hph gene during meiotic segregation. The NIILs (BC(2)F(2)) showed maximum similarity with the recurrent parent cultivar IR64. Further, progenies of two NIILs were devoid of any fragments beyond the left or right border, including the chloramphenicol acetyltransferase (cat) antibiotic resistance gene of the transformation vector. Spectrophotometric readings showed the accumulation of up to 1.06 microg total carotenoids, including beta-carotene, in 1 g of the endosperm. The accumulation of beta-carotene was also evident from the clearly visible yellow colour of the polished seeds. PMID:17177811
Baisakh, Niranjan; Rehana, Sayda; Rai, Mayank; Oliva, Norman; Tan, Jing; Mackill, David J; Khush, Gurdev S; Datta, Karabi; Datta, Swapan K
Over-expression of snakin-2 and extensin-like protein genes restricts pathogen invasiveness and enhances tolerance to Clavibacter michiganensis subsp. michiganensis in transgenic tomato (Solanum lycopersicum).
Two tomato proteins were evaluated by over-expression in transgenic tomato for their ability to confer resistance to Clavibacter michiganensis subsp. michiganensis (Cmm). Snakin-2 (SN2) is a cysteine-rich peptide with broad-spectrum antimicrobial activity in vitro while extensin-like protein (ELP) is a major cell-wall hydroxyproline-rich glycoprotein linked with plant response to pathogen attack and wounding. Tomato plants, cultivar Mountain Fresh, were transformed via Agrobacterium tumefaciens harboring a binary vector for expression of the full-length SN2 gene or ELP cDNA under the regulation of the CaMV 35S promoter. Molecular characterization of PCR-positive putative T(0) transgenic plants by Northern analysis revealed constitutive over-expression of SN2 and ELP mRNA. Junction fragment analysis by Southern blot showed that three of the four SN2 over-expressing T(0) lines had single copies of complete T-DNAs while the other line had two complete T-DNA copies. All four ELP over-expressing T(0) lines had a single copy T-DNA insertion. Semi-quantitative RT-PCR analysis of T(1) plants revealed constitutive over-expression of SN2 and ELP. Transgenic lines that accumulated high levels of SN2 or ELP mRNA showed enhanced tolerance to Cmm resulting in a significant delay in the development of wilt symptoms and a reduction in the size of canker lesions compared to non-transformed control plants. Furthermore, in transgenic lines over-expressing SN2 or ELP bacterial populations were significantly lower (100-10,000-fold) than in non-transformed control plants. These results demonstrate that SN2 and ELP over-expression limits Cmm invasiveness suggesting potential in vivo antibacterial activity and possible biotechnological application for these two defense proteins. PMID:21479554
Balaji, Vasudevan; Smart, Christine D
5Azacytidine prevents transgene methylation in vivo
Retroviral sequence can silence transgene expression in vitro and in vivo. We report that this effect can be efficiently prevented by in vivo administration of the demethylating agent 5-azacytidine (aza-C). We engineered the U937 human cell line with a retroviral vector consisting of the thymidine kinase suicide gene (tk), which induces sensitivity to ganciclovir (gcv) and through an IRES sequence,
M Di Ianni; A Terenzi; K Perruccio; R Ciurnelli; F Lucheroni; R Benedetti; M F Martelli; A Tabilio
Recombinant protein production in transgenic animals
The engineering of animals for recombinant protein production has gone beyond the stage of identifying proper regulatory sequences. Efforts are now spent on the generation of transgenic animals that process heterologous proteins more efficiently. Another line of research is the development of strategies aimed at bypassing pronuclear microinjection.
Yann Echelard
Two distinct determinants of ligand specificity in T1R1/T1R3 (the umami taste receptor).
Umami taste perception in mammals is mediated by a heteromeric complex of two G-protein-coupled receptors, T1R1 and T1R3. T1R1/T1R3 exhibits species-dependent differences in ligand specificity; human T1R1/T1R3 specifically responds to L-Glu, whereas mouse T1R1/T1R3 responds more strongly to other L-amino acids than to L-Glu. The mechanism underlying this species difference remains unknown. In this study we analyzed chimeric human-mouse receptors and point mutants of T1R1/T1R3 and identified 12 key residues that modulate amino acid recognition in the human- and mouse-type responses in the extracellular Venus flytrap domain of T1R1. Molecular modeling revealed that the residues critical for human-type acidic amino acid recognition were located at the orthosteric ligand binding site. In contrast, all of the key residues for the mouse-type broad response were located at regions outside of both the orthosteric ligand binding site and the allosteric binding site for inosine-5'-monophosphate (IMP), a known natural umami taste enhancer. Site-directed mutagenesis demonstrated that the newly identified key residues for the mouse-type responses modulated receptor activity in a manner distinct from that of the allosteric modulation via IMP. Analyses of multiple point mutants suggested that the combination of two distinct determinants, amino acid selectivity at the orthosteric site and receptor activity modulation at the non-orthosteric sites, may mediate the ligand specificity of T1R1/T1R3. This hypothesis was supported by the results of studies using nonhuman primate T1R1 receptors. A complex molecular mechanism involving changes in the properties of both the orthosteric and non-orthosteric sites of T1R1 underlies the determination of ligand specificity in mammalian T1R1/T1R3. PMID:24214976
Toda, Yasuka; Nakagita, Tomoya; Hayakawa, Takashi; Okada, Shinji; Narukawa, Masataka; Imai, Hiroo; Ishimaru, Yoshiro; Misaka, Takumi
Live Astrocytes Visualized by Green Fluorescent Protein in Transgenic Mice
Green fluorescent protein (hGFP-S65T) was expressed in transgenic mice under the control of the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter. Tissues from two independent transgenic lines were characterized by Northern blot analysis and by confocal microscopy. The expression pattern in these two lines was identical in all tissues examined, and similar to that found previously with alacZtransgene driven by
Lang Zhuo; Biao Sun; Chuan-Li Zhang; Alan Fine; Shing-Yan Chiu; Albee Messing
Simultaneous confirmatory analysis of different transgenic maize (zea mays) lines using multiplex polymerase chain reaction-restriction analysis and capillary gel electrophoresis with laser induced fluorescence detection.
A novel analytical procedure based on the combination of multiplex PCR, restriction analysis, and CGE-LIF to unambiguosly and simultaneously confirm the presence of multiple lines of genetically modified corn is proposed. This methodology is based on the amplification of event-specific DNA regions by multiplex PCR using 6-FAM-labeled primers. Subsequently, PCR products are digested by a mixture containing specific restriction endonucleases. Thus, restriction endonucleases selectively recognize DNA target sequences contained in the PCR products and cleave the double-stranded DNA at a given cleavage site. Next, the restriction digest is analyzed by CGE-LIF corroborating the length of the expected restriction fragments, confirming (or not) the existence of GMOs. For accurate size determination of the DNA fragments by CGE-LIF a special standard DNA mixture was produced in this laboratory for calibration. The suitability of this mixture for size determination of labeled DNA fragments is also demonstrated. The usefulness of the proposed methodology is demonstrated through the simultaneous detection and confirmatory analysis of samples containing 0.5% of GA21 and MON863 maize plus an endogenous gene of maize as control. PMID:18710253
GarcĒa-Ca±as, Virginia; Cifuentes, Alejandro
T1-mapping in the heart: accuracy and precision
The longitudinal relaxation time constant (T1) of the myocardium is altered in various disease states due to increased water content or other changes to the local molecular environment. Changes in both native T1 and T1 following administration of gadolinium (Gd) based contrast agents are considered important biomarkers and multiple methods have been suggested for quantifying myocardial T1 in vivo. Characterization of the native T1 of myocardial tissue may be used to detect and assess various cardiomyopathies while measurement of T1 with extracellular Gd based contrast agents provides additional information about the extracellular volume (ECV) fraction. The latter is particularly valuable for more diffuse diseases that are more challenging to detect using conventional late gadolinium enhancement (LGE). Both T1 and ECV measures have been shown to have important prognostic significance. T1-mapping has the potential to detect and quantify diffuse fibrosis at an early stage provided that the measurements have adequate reproducibility. Inversion recovery methods such as MOLLI have excellent precision and are highly reproducible when using tightly controlled protocols. The MOLLI method is widely available and is relatively mature. The accuracy of inversion recovery techniques is affected significantly by magnetization transfer (MT). Despite this, the estimate of apparent T1 using inversion recovery is a sensitive measure, which has been demonstrated to be a useful tool in characterizing tissue and discriminating disease. Saturation recovery methods have the potential to provide a more accurate measurement of T1 that is less sensitive to MT as well as other factors. Saturation recovery techniques are, however, noisier and somewhat more artifact prone and have not demonstrated the same level of reproducibility at this point in time. This review article focuses on the technical aspects of key T1-mapping methods and imaging protocols and describes their limitations including the factors that influence their accuracy, precision, and reproducibility.
Spatially regularized T1 estimation from variable flip angles MRI
Purpose: To develop efficient algorithms for fast voxel-by-voxel quantification of tissue longitudinal relaxation time (T1) from variable flip angles magnetic resonance images (MRI) to reduce voxel-level noise without blurring tissue edges. Methods: T1 estimations regularized by total variation (TV) and quadratic penalty are developed to measure T1 from fast variable flip angles MRI and to reduce voxel-level noise without decreasing the accuracy of the estimates. First, a quadratic surrogate for a log likelihood cost function of T1 estimation is derived based upon the majorization principle, and then the TV-regularized surrogate function is optimized by the fast iterative shrinkage thresholding algorithm. A fast optimization algorithm for the quadratically regularized T1 estimation is also presented. The proposed methods are evaluated by the simulated and experimental MR data. Results: The means of the T1 values in the simulated brain data estimated by the conventional, TV-regularized, and quadratically regularized methods have less than 3% error from the true T1 in both GM and WM tissues with image noise up to 9%. The relative standard deviations (SDs) of the T1 values estimated by the conventional method are more than 12% and 15% when the images have 7% and 9% noise, respectively. In comparison, the TV-regularized and quadratically regularized methods are able to suppress the relative SDs of the estimated T1 to be less than 2% and 3%, respectively, regardless of the image noise level. However, the quadratically regularized method tends to overblur the edges compared to the TV-regularized method. Conclusions: The spatially regularized methods improve quality of T1 estimation from multiflip angles MRI. Quantification of dynamic contrast-enhanced MRI can benefit from the high quality measurement of native T1.
Wang, Hesheng; Cao, Yue
Irreversible change in the T1 temperature dependence with thermal dose using the PRF-T1 Technique
Denaturation of macromolecules within the tissues is believed to be the major factor contributing to the damage of tissues upon hyperthermia. As a result, the value of the spin-lattice relaxation time T1 of the tissue water, which is related to the translational and rotational rates of water, represents an intrinsic probe for investigating structural changes in tissues at high temperature. Therefore, the goal of the present work is to investigate whether the simultaneous measurement of temperature and T1 using a hybrid PRF-T1 measurement technique, can be used to detect irreversible changes in T1 that might be indicative of tissue damage. A new hybrid PRF-T1 sequence was implemented based on the variable flip angle DESPOT1 method from a standard 3D segmented EPI sequence by alternating two flip angles from measurement to measurement. The structural changes of the heated tissue volumes were analyzed based on the derived T1 values and the corresponding PRF temperatures. Using the hybrid PRF-T1 technique, we demonstrate that the change of spin lattice relaxation time T1 is reversible with temperature for low thermal dose (thermal dose ? 240 CEM43░C) and irreversible with temperature after significant accumulation of thermal dose in ex vivo chicken breast tissue. These results suggest that the hybrid PRF-T1 method may be a potentially powerful tool to investigate the extent and mechanism of heat damage of biological tissues.
Diakite, Mahamadou; Payne, Allison; Todd, Nick; Parker, Dennis L.
A triple-transgenic immunotolerant mouse model.
Avoiding unwanted immunogenicity is of key importance in the development of therapeutic drug proteins. Animal models are of less predictive value because most of the drug proteins are recognized as foreign proteins. However, different methods have been developed to obtain immunotolerant animal models. So far, the immunotolerant animal models have been developed to assess one protein at a time and are not suitable for the assessment of combination products. Our aim was to develop an animal model for evaluating the impact of manufacturing and formulation changes on immunogenicity, suitable for both single protein and combination products. We constructed two lines of transgenic mice expressing the three human coagulation factors, II, VII, and X, by inserting a single vector containing the three coagulation factors encoding sequences separated by insulator sequences derived from the chicken beta-globin locus into the mouse genome. Immunization of transgenic mice from the two lines and their wild-type littermates showed that transgenic mice from both lines were immunotolerant to the expressed human coagulation factors. We conclude that transgenic mice immunotolerant to multiple proteins can be obtained, and that these mice are potentially useful as animal models in the assessment of immunogenicity in response to manufacturing changes. PMID:23316010
Brenden, Nina; Madeyski-Bengtson, Katja; Martinsson, Klara; Svõrd, Rebecka; Albery-Larsdotter, Sara; Granath, Britta; Lundgren, Hanna; L÷vgren, Ann
Transgenic Gladiolus plants transformed with the bean yellow mosaic virus coat-protein gene in either sense or antisense orientation.
Transgenic Gladiolus plants transformed with the bean yellow mosaic virus (BYMV) coat-protein (CP) gene in either sense or antisense (AS) orientation were developed using biolistics. Four of the plants were confirmed to carry the CP gene in the sense orientation of the gene and seven plants in the AS orientation. Two of the CP plant lines and all of the AS lines showed DNA rearrangements of the transgene in addition to an intact copy of the transgene. The copy number ranged from one to nine. Of the 11 lines, eight had only one to four copies of the transgene. Transcription of the transgene occurred for three of the CP lines and five of the AS lines as determined by Northern hybridization. All 11 plant lines were challenged with BYMV using controlled aphid transmission. One month following aphid transmission, the transgenic plants were examined by immunoelectron microscopy for presence of the virus. Several transgenic plant lines containing either antiviral transgene showed a lower incidence of infection (percentage of plants infected as detected by immunoelectron microscopy) than the non-transformed plants. Most of the CP- and AS-transgenic plants that did not contain BYMV 1 month after challenge were found to contain BYMV the next season. It appeared that BYMV infection was delayed in the CP- and AS-transgenic lines but that the transgenes did not prevent eventual infection of BYMV. This is the first report of developing a floral bulb crop with antiviral genes to BYMV. PMID:15480682
Kamo, Kathryn; Gera, Abed; Cohen, Jacob; Hammond, John; Blowers, Alan; Smith, Franzine; Van Eck, Joyce
Transgenic mice as research tools in neurocarcinogenesis.
Transgenic animal models for neurocarcinogenesis have provided significant insights into the molecular mechanisms underlying carcinogenic processes, including those which affect the nervous system. In view of the very rapid pace of acquisition of knowledge, it is not possible to cover all transgenic mouse models for neural tumors. Instead, this article discusses some of the most important technical innovations for manipulation of the mammalian genome (notably the various methods for targeted genome modifications, as well as the technology for introducing large DNA fragments into the germ line of mice), and presents a selection of the transgenic mouse models which are proving most promising for furthering our understanding of the pathogenetic basis of cancer in the nervous system. PMID:9584953
Rovigatti, U; Afanasyeva, T; Brandner, S; Hainfellner, J A; Kiess, M; Maddalena, A; Malin, G; R³licke, T; Steinbach, J; Weissenberger, J; Aguzzi, A
Expression of spearmint limonene synthase in transgenic spike lavender results in an altered monoterpene composition in developing leaves.
We generated transgenic spike lavender (Lavandula latifolia) plants constitutively expressing the limonene synthase (LS) gene from spearmint (Mentha spicata), encoding the LS enzyme that catalyzes the synthesis of limonene from geranyl diphosphate. Overexpression of the LS transgene did not consistently affect monoterpene profile in pooled leaves or flowers from transgenic T(0) plants. Analyses from cohorts of leaves sampled at different developmental stages showed that essential oil accumulation in transgenic and control plants was higher in developing than in mature leaves. Furthermore, developing leaves of transgenic plants contained increased limonene contents (more than 450% increase compared to controls) that correlated with the highest transcript accumulation of the LS gene. The levels of other monoterpene pathway components were also significantly altered. T(0) transgenic plants were grown for 2 years, self-pollinated, and the T(1) seeds obtained. The increased limonene phenotype was maintained in the progenies that inherited the LS transgene. PMID:18514005
Mu±oz-Bertomeu, Jes·s; Ros, Roc; Arrillaga, Isabel; Segura, Juan
The production of recombinant proteins is one of the major successes of biotechnology. Animal cells are required to synthesize\\u000a proteins with the appropriate post-translational modifications. Transgenic animals are being used for this purpose. Milk,\\u000a egg white, blood, urine, seminal plasma and silk worm cocoon from transgenic animals are candidates to be the source of recombinant\\u000a proteins at an industrial scale.
Louis Marie Houdebine
Accumulation of transgene-derived siRNAs is not sufficient for RNAi-mediated protection against Citrus tristeza virus in transgenic Mexican lime.
Mexican lime plants transformed with the 3'-terminal 549 nucleotides of the Citrus tristeza virus (CTV) genome in sense, antisense and intron-hairpin formats were analysed for transgene-derived transcript and short interfering RNA (siRNA) accumulation, and for CTV resistance. Propagations from all sense, antisense and empty-vector transgenic lines were susceptible to CTV, except for a single sense-line plant with a complex transgene integration pattern that showed transgene-derived siRNAs in association with low levels of the transgene-derived transcript. In contrast, nine of 30 intron-hairpin lines showed CTV resistance, with 9%-56% of bud-propagated plants, depending on the line, remaining uninfected on graft inoculation, and the others being susceptible. Although resistance was always associated with the presence of transgene-derived siRNAs, their level in different sense and intron-hairpin transformants was variable irrespective of the response to CTV infection. In intron-hairpin lines with single transgene integration, CTV resistance was correlated with low accumulation of the transgene-derived transcript rather than with high accumulation of transgene-derived siRNAs. PMID:20078774
L¾pez, Carmelo; Cervera, Magdalena; Fagoaga, Carmen; Moreno, Pedro; Navarro, Luis; Flores, Ricardo; Pe±a, Leandro
Improving organic phosphate utilization in transgenic white clover by overexpression of Aspergillus niger PhyA gene
Using the cotyledon of white clover as explants, the transgenic white clover lines ectopic expression of the PhyA gene were established based on Agrobacterium tumefaciens-mediated transformation method. It was found that the tested transgenic lines were all Polymerase Chain Reaction (PCR) positive.\\u000a The transgenic lines 1 to 4 were used for further Southern blot and Northern blot analysis. The lines
Shengfang Han; Juntao Gu; Kai Xiao
Suppression of Virus Accumulation in Transgenic Plants Exhibiting Silencing of Nuclear Genes.
Homology-dependent gene silencing contributes to variation between transgenic plants with respect to transgene and/or endogenous gene expression levels. Recent studies have linked post-transcriptional gene silencing and virus resistance in plants expressing virus-derived transgenes. Using a potato virus X vector, we present three examples in which silencing of nonviral transgenes prevented virus accumulation. This effect was dependent on sequence homology between the virus and the silenced transgene. Analysis of potato virus X derivatives carrying bacterial [beta]-glucuronidase (GUS) sequences showed that the 3[prime] region of the GUS coding sequence was a target of the silencing mechanism in two independent tobacco lines. Methylation of the silenced GUS transgenes in these lines was also concentrated in the 3[prime] region of the GUS coding sequence. Based on this concurrence, we propose a link between the DNA-based transgene methylation and the RNA-based gene silencing process.
English, J. J.; Mueller, E.; Baulcombe, D. C.
Resistance levels and fitness of protoporphyrinogen oxidase (PROTOX) inhibitor-resistant transgenic rice in paddy fields
We previously confirmed that the transgenic rice line, M4, was about 200-fold more resistant to oxyfluorfen than the wild-type (WT) rice in whole-plant bioassays in pots. The transgenic rice line was also cross-resistant to other protoporphyrinogen oxidase (PROTOX)-inhibiting herbicides, acifluorfen, carfentrazone, and oxadiazon. The objectives of this research were to (a) verify the resistance of transgenic rice plants to commercial
H. I. Jung; Y. I. Kuk; H. Y. Kim; K. Back; D. J. Lee; S. Lee; N. R. Burgos
A thermoalkaliphilic lipase of Geobacillus sp. T1
A thermoalkaliphilic T1 lipase gene of Geobacillus sp. strain T1 was overexpressed in pGEX vector in the prokaryotic system. Removal of the signal peptide improved protein\\u000a solubility and promoted the binding of GST moiety to the glutathione-Sepharose column. High-yield purification of T1 lipase\\u000a was achieved through two-step affinity chromatography with a final specific activity and yield of 958.2ĀU\\/mg and 51.5%,
Thean Chor Leow; Raja Noor Zaliha Raja Abd Rahman; Mahiran Basri; Abu Bakar Salleh
Dealing with a New T1D Diagnosis in College
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Irreversible change in the T1 temperature dependence with thermal dose using the proton resonance frequency-T1 technique.
Denaturation of macromolecules within the tissues is believed to be the major factor contributing to the damage of tissues upon hyperthermia. As a result, the value of the spin-lattice relaxation time T1 of the tissue water, which is related to the translational and rotational rates of water, represents an intrinsic probe for investigating structural changes in tissues at high temperature. Therefore, the goal of this work is to investigate whether the simultaneous measurement of temperature and T1 using a hybrid proton resonance frequency (PRF)-T1 measurement technique can be used to detect irreversible changes in T1 that might be indicative of tissue damage. A new hybrid PRF-T1 sequence was implemented based on the variable flip angle driven-equilibrium single-pulse observation (DESPOT)1 method from a standard three dimensional segmented echo-planar imaging sequence by alternating two flip angles from measurement to measurement. The structural changes of the heated tissue volumes were analyzed based on the derived T1 values and the corresponding PRF temperatures. Using the hybrid PRF-T1 technique, we demonstrate that the change of spin lattice relaxation time T1 is reversible with temperature for low thermal dose (thermal dose ? 240 cumulative equivalent minutes [CEM] 43░C) and irreversible with temperature after significant accumulation of thermal dose in ex vivo chicken breast tissue. These results suggest that the hybrid PRF-T1 method may be a potentially powerful tool to investigate the extent and mechanism of heat damage of biological tissues. PMID:22576265
Diakite, Mahamadou; Payne, Allison; Todd, Nick; Parker, Dennis L
T1C photometry of NGC7507 (Caso+, 2013)
NASA Astrophysics Data System (ADS)
We present a catalog of point-like sources around NGC 7507, which forms the photometric database for our paper. The sources were obtained from the PSF photometry of MOSAIC images in filters R and C. The catalog contains coordinates, T1 magnitudes with uncertainties, and C-T1 colors and their uncertainties. Magnitudes and colour are corrected by absorption and reddening. (1 data file).
Caso, J. P.; Richtler, T.; Bassino, L. P.; Salinas, R.; Lane, R. R.; Romanowsky, A.
T-cell receptor transgenic mice in the study of autoimmune diseases
T cell receptor transgenic mice have been a valuable tool in the study of the immune system, from development to selection to tolerance or pathogenesis. In this manuscript we review the T cell receptor transgenic mouse lines with specificity for self antigens that have been reported before August 2003. Many such lines have been generated, which have been instrumental in
Juan J Lafaille
Recent advances in the development of new transgenic animal technology.
Transgenic animal technology is one of the fastest growing biotechnology areas. It is used to integrate exogenous genes into the animal genome by genetic engineering technology so that these genes can be inherited and expressed by offspring. The transgenic efficiency and precise control of gene expression are the key limiting factors in the production of transgenic animals. A variety of transgenic technologies are available. Each has its own advantages and disadvantages and needs further study because of unresolved technical and safety issues. Further studies will allow transgenic technology to explore gene function, animal genetic improvement, bioreactors, animal disease models, and organ transplantation. This article reviews the recently developed animal transgenic technologies, including the germ line stem cell-mediated method to improve efficiency, gene targeting to improve accuracy, RNA interference-mediated gene silencing technology, zinc-finger nuclease gene targeting technology and induced pluripotent stem cell technology. These new transgenic techniques can provide a better platform to develop transgenic animals for breeding new animal varieties and promote the development of medical sciences, livestock production, and other fields. PMID:22833168
Miao, Xiangyang
A T1-based DSP modem for interfacing voice and packet networks
A packet-switched data network (PSDN) with dial-in or dial-out capability requires a large number of interfaces to the public switched telephone network (PSTN). A T1 trunk connected to an X.25 trunk by a multiple digital signal processing modem is proposed for this interface in place of individual telephone lines, modems and computer cables. The design and performance for this type
H. E. White
Mouse transgenic approaches in optogenetics
A major challenge in neuroscience is to understand how universal behaviors, such as sensation, movement, cognition, and emotion, arise from the interactions of specific cells that are present within intricate neural networks in the brain. Dissection of such complex networks has typically relied on disturbing the activity of individual gene products, perturbing neuronal activities pharmacologically, or lesioning specific brain regions, to investigate the networkÆs response in a behavioral output. Though informative for many kinds of studies, these approaches are not sufficiently fine-tuned for examining the functionality of specific cells or cell classes in a spatially or temporally-restricted context. Recent advances in the field of optogenetics now enable researchers to monitor and manipulate the activity of genetically defined cell populations with the speed and precision uniquely afforded by light. Transgenic mice engineered to express optogenetic tools in a cell type-specific manner offer a powerful approach for examining the role of particular cells in discrete circuits in a defined and reproducible way. Not surprisingly then, recent years have seen substantial efforts directed towards generating transgenic mouse lines that express functionally relevant levels of optogenetic tools. In this chapter, we review the state of these efforts and consider aspects of the current technology that would benefit from additional improvement.
Zeng, Hongkui; Madisen, Linda
Analysis of T-DNA- Xa21 loci and bacterial blight resistance effects of the transgene Xa21 in transgenic rice
The genetic loci and phenotypic effects of the transgene Xa21, a bacterial blight (BB) resistance gene cloned from rice, were investigated in transgenic rice produced through an Agrobacterium-mediated transformation system. The flanking sequences of integrated T-DNAs were isolated from Xa21 transgenic rice lines using thermal asymmetric interlaced PCR. Based on the analysis of 24 T-DNA- Xa21 flanking sequences, T-DNA loci
Wenxue Zhai; Caiyan Chen; Xuefeng Zhu; Xuewei Chen; Dechun Zhang; Xiaobing Li; Lihuang Zhu
Regeneration of transgenic citrus plants under non selective conditions results in high-frequency recovery of plants with silenced transgenes.
Insertion of foreign DNA into plant genomes frequently results in the recovery of transgenic plants with silenced transgenes. To investigate to what extent regeneration under selective conditions limits the recovery of transgenic plants showing gene silencing in woody species, Mexican lime [ Citrus aurantifolia (Christm.) Swing.] plants were transformed with the p25 coat protein gene of Citrus tristeza virus (CTV) with or without selection for nptII and uidA. Strikingly, more than 30% of the transgenic limes regenerated under non-selective conditions had silenced transgenes, and in all cases silencing affected all the three transgenes incorporated. These results indicate that the frequency of transgene silencing may be greatly underestimated when the rate of silencing is estimated from the number of regenerants obtained under selective conditions. To our knowledge, this is the first report in which the frequency of gene silencing after transformation has been quantified. When the integration pattern of T-DNA was analyzed in silenced and non-silenced lines, it was observed that inverted repeats as well as direct repeats and even single integrations were able to trigger gene silencing. Gene silencing has often been associated with the insertion of DNA sequences as inverted repeats. Interestingly, here, direct repeats and single-copy insertions were found in both silenced and non-silenced lines, suggesting that the presence of inverted-repeat T-DNAs and the subsequent formation of dsRNAs triggering gene silencing cannot account for all silencing events. PMID:12111562
DomĒnguez, A; Fagoaga, C; Navarro, L; Moreno, P; Pe±a, L
Global Regulation of Transgenic Crops
Globally, transgenic maize comprised about 25% of the 102 million hectares of transgenic cropland planted in 2006 by more\\u000a than 10 million farmers in 21 countries (James 2007). These transgenic maize plants contain inserted gene(s) expressing a\\u000a variety of Cry proteins that confer resistance to stem borers and rootworms. Approximately 45% of the transgenic maize planted\\u000a also contains inserted gene(s)
Bruce M. Chassy
Elastin gene mutations in transgenic mice.
We have constructed several rat tropoelastin minigene recombinants encoding the complete sequence of rat tropoelastin, two isoforms of rat tropoelastin and a truncated tropoelastin lacking the domains encoded by exons 19-31 of the rat gene. Coding and non-coding domains in all these recombinants were placed under the transcriptional control of 3 kb of the promoter domain of the rat tropoelastin gene. These minigenes were used to prepare a total of 28 separate founder lines of transgenic mice. A species-specific reverse-transcriptase polymerase chain reaction (RT-PCR) assay was established to demonstrate the synthesis of rat and mouse tropoelastin mRNA in several tissues obtained from both neonatal and adult transgenic mice. Thermolytic digestion of insoluble elastin isolated from several neonatal mouse tissues revealed the presence of rat tropoelastin peptides in progeny from all those founder mice in which detectable levels of rat tropoelastin mRNA were noted. Phenotypic and histopathological assessment of transgenic and non-transgenic animals revealed the development of two diverse elastic tissue disorders. The progeny of two separate founder lines overexpressing the rat tropoelastin isoform lacking exon 33, developed an emphysematous phenotype in early adulthood. In contrast, transgenic mice, in which expression of the truncated rat tropoelastin minigene lacking exons 19-31 had been observed, died of a ruptured ascending aortic aneurysm. Tropoelastin gene mutations, therefore, will result in heritable disorders of elastic tissue. Moreover, different mutations in the tropoelastin gene will be responsible for very different abnormalities in elastic tissue function. PMID:8575255
Sechler, J L; Sandberg, L B; Roos, P J; Snyder, I; Amenta, P S; Riley, D J; Boyd, C D
Responses of MxPPO overexpressing transgenic tall fescue plants to two diphenyl-ether herbicides, oxyfluorfen and acifluorfen
We generated transgenic tall fescue (Festuca arundinacea Schreb. cv. Kentucky-31) plants harboring a synthetic Myxococcus xanthus protoporphyrinogen oxidase (MxPPO) gene through Agrobacterium-mediated gene transfer. Successful integration of the transgene into the genome of transgenic plants confirmed by polymerase\\u000a chain reaction (PCR) and Southern blot analysis, and the functional expression of the MxPPO gene at the mRNA level in transgenic lines
Ki-Won Lee; Nagib Ahsan; Sang-Hoon Lee; Dong-Gi Lee; Kyung-Hee Kim; Iftekhar Alam; Suk-Yoon Kwon; Jin-Seog Kim; Kyoungwhan Back; Sung Sil Lee; Byung-Hyun Lee
Generation of transgenic papaya with double resistance to Papaya ringspot virus and Papaya leaf-distortion mosaic virus.
During the field tests of coat protein (CP)-transgenic papaya lines resistant to Papaya ringspot virus (PRSV), another Potyvirus sp., Papaya leaf-distortion mosaic virus (PLDMV), appeared as an emerging threat to the transgenic papaya. In this investigation, an untranslatable chimeric construct containing the truncated CP coding region of the PLDMV P-TW-WF isolate and the truncated CP coding region with the complete 3' untranslated region of PRSV YK isolate was transferred into papaya (Carica papaya cv. Thailand) via Agrobacterium-mediated transformation to generate transgenic plants with resistance to PLDMV and PRSV. Seventy-five transgenic lines were obtained and challenged with PRSV YK or PLDMV P-TW-WF by mechanical inoculation under greenhouse conditions. Thirty-eight transgenic lines showing no symptoms 1 month after inoculation were regarded as highly resistant lines. Southern and Northern analyses revealed that four weakly resistant lines have one or two inserts of the construct and accumulate detectable amounts of transgene transcript, whereas nine resistant lines contain two or three inserts without significant accumulation of transgene transcript. The results indicated that double virus resistance in transgenic lines resulted from double or more copies of the insert through the mechanism of RNA-mediated posttranscriptional gene silencing. Furthermore, three of nine resistant lines showed high levels of resistance to heterologous PRSV strains originating from Hawaii, Thailand, and Mexico. Our transgenic lines have great potential for controlling a number of PRSV strains and PLDMV in Taiwan and elsewhere. PMID:19821736
Kung, Yi-Jung; Bau, Huey-Jiunn; Wu, Yi-Ling; Huang, Chiung-Huei; Chen, Tsui-Miao; Yeh, Shyi-Dong
Overexpression of growth-associated proteins in the neurons of adult transgenic mice.
The Thy-1.2 expression cassette described in this report drives strong constitutive expression of transgenes specifically in the neurons of adult transgenic mice. Transgene expression begins at P6-10, at a time when it can affect activity-dependent rearrangements of synaptic connections and neuron-glia interactions during the late phases of nervous system development. Slightly earlier expression is detected in the developing cerebellum, where transgenes could also affect granule cell migration. Probably due to the strong genetic context sensitivity of the expression construct, expression patterns differ considerably among transgenic lines. Accordingly, neuron types can be subdivided into a group that consistently displays substantial expression in all tested transgenic lines, and in those where expression is founder-specific. About a fifth of the transgenic lines displayed quite generalized expression in neurons. These properties of the Thy-1.2 expression cassette can be exploited to study the effects of transgenes during late nervous system development and in the adult, with the added opportunity of analyzing different combinations of expressing and non-expressing neurons in transgenic animals derived from different founder lines. PMID:9125370
Caroni, P
Myocardial T1 mapping: modalities and clinical applications
Myocardial fibrosis appears to be linked to myocardial dysfunction in a multitude of non-ischemic cardiomyopathies. Accurate non-invasive quantitation of this extra-cellular matrix has the potential for widespread clinical benefit in both diagnosis and guiding therapeutic intervention. T1 mapping is a cardiac magnetic resonance (CMR) imaging technique, which shows early clinical promise particularly in the setting of diffuse fibrosis. This review will outline the evolution of T1 mapping and the various techniques available with their inherent advantages and limitations. Histological validation of this technique remains somewhat limited, however clinical application in a range of pathologies suggests strong potential for future development.
Jellis, Christine L.
[pT1G3 bladder carcinoma: our experience].
Approximately 6 to 23% of the patients with transitional cell carcinoma confined to the superficial mucosa of the bladder suffer for a pT1G3 tumor. Between 1984 and 1994, 12 patients were treated for high grade stage T1 tumores. Trans-urethral resection of the bladder cancer was performed in 8 patients, supported in two cases by immunotherapy with B.C.G. and in one case by endovesical chemotherapy. Radical cystectomy was carried out in 4 patients. Our results are similar to what reported by other Authors, but we didn't have any progression in all 8 patients treated with conservative therapy. PMID:8664927
Dell'Orto, P; Trinchieri, A; D'Addezio, F; Bernardini, P; Mangiarotti, B; Del Nero, A; Pisani, E
Efficacy and mechanism of action of Deguelin in suppressing metastasis of 4T1 cells.
Cancer related deaths in breast cancer patients are due to metastasis of the disease. Murine 4T1 cells (Murine mammary cancer cell line developed from 6-thioguanine resistant tumor) provide an excellent research tool for metastasis related studies because these cells are highly aggressive and readily metastasize to the lungs. In this study we determined the effect of Deguelin on in vivo/vitro growth and metastasis of 4T1 cells. Deguelin inhibited the in vitro growth of 4T1 cells in a time and dose dependent manner accompanied with reduced nuclear PCNA immunostaining. In cells treated with Deguelin, reduced expression of nuclear c-Met, and its downstream targets such p-ERK and p-AKT was observed. Deguelin reduced the cell migration in 4T1 cells as determined by scratch wound assay. Combined treatment with DeguelinĀ+ĀERK or PI3K/AKT inhibitor had no additional effect on cell migration. These results indicated that the action of Deguelin on cell migration may be mediated by AKT and ERK mediated signaling pathways. In vivo, Deguelin treatment significantly inhibited growth of 4T1 cells. Deguelin also reduced the occurrence of metastatic lung lesions by 33Ā% when cells were injected intravenously into Balb/c female mice. There was no difference in the body weight, nor was there a difference in liver and spleen weights between vehicle treated-control and Deguelin-treated animals, which indicated that Deguelin was nontoxic at the dose used in the present study. These results provide rationale for developing Deguelin as a chemotherapeutic agent for triple negative breast cancer patients. PMID:23645347
Mehta, Rajeshwari R; Katta, Harshadadevi; Kalra, Amit; Patel, Rutulkumar; Gupta, Akash; Alimirah, Fatouma; Murillo, Genoveva; Peng, Xinjian; Unni, Aditya; Muzzio, Miguel; Mehta, Rajendra G
Transfer of hyperpolarization from long T 1 storage nuclei to short T 1 neighbors using FLOPSY-8
NASA Astrophysics Data System (ADS)
Nuclei with long T 1s are optimal targets for dynamic nuclear polarization (DNP). Therefore, most of the agents used in metabolic imaging and spectroscopy studies are based on carboxylic acid moieties that lack protons, a strong source of dipolar relaxation. Metabolic flux information encoded into spectra of small molecule metabolites in the form of the 13C isotopomer data cannot be accessed using standard 13C hyperpolarization methods because protonated carbons relax too quickly through T 1 dipolar relaxation. It is shown here that the longitudinal mixing sequence FLOPSY-8 can be used to transfer polarization from a long T 1 storage nucleus to adjacent protonated carbons so that they may be detected with high sensitivity. We demonstrate that FLOPSY-8 allows a direct readout of isotopomer populations in butyrate and glutamate in vitro.
Moreno, Karlos X.; Harrison, Crystal; Dean Sherry, A.; Malloy, Craig R.; Merritt, Matthew E.
Effect of Lymphatic Mapping on Diagnosis and Treatment of Patients With T1a, T1b Favorable Breast Cancer
Objective To investigate the incidence of nodal metastasis in a consecutive series of patients treated at the authorsÆ institution with highly selective criteria, and to determine the impact that lymphatic mapping and sentinel node biopsy have on the detection of nodal metastases in this carefully selected patient population. Methods Study patients were selected from the 7,750 breast cancer patients entered into the authorsÆ database from April 1989 to August 2001, based on the following criteria: nonpalpable, T1a and T1b, non-high nuclear grade tumors, without lymphovascular invasion. Results Of the 7,750 patients in the database 1,327 (17%) were found to have T1a and T1b lesions. Three hundred eighty-nine patients were confirmed to meet all four selection criteria. This represents 5% (389/7,750) of the authorsÆ breast cancer patients and 29.3% (389/1,327) of the authorsÆ T1a/T1b tumors. One hundred sixty patients were diagnosed before routine use of lymphatic mapping, and only one patient had a positive axillary lymph node. Two hundred twenty-nine patients underwent lymphatic mapping and sentinel lymph node biopsy, and 10 had a positive axillary lymph node. The difference in proportions of nodal positivity between the mapped and unmapped patients was significant. Conclusions This study clearly demonstrates the ability of lymphatic mapping and a more detailed examination of the sentinel node to increase the accuracy of axillary staging. It has been argued that this highly selected group of breast cancer patients possessing retrospectively identified ōfavorableö characteristics does not require axillary staging. This select population represents only 5% of breast cancer patients in this series, and the authors do not believe they can be accurately identified preoperatively. Therefore, the authors strongly argue for evaluation of the axillary nodal status by lymphatic mapping.
Jakub, James W.; Ebert, Mark D.; Diaz, Nils M.; Cantor, Alan; Reintgen, Douglas S.; Dupont, Elisabeth L.; Shons, Alan R.; Cox, Charles E.
Resistance pattern and antioxidant enzyme profiles of protoporphyrinogen oxidase (PROTOX) inhibitor-resistant transgenic rice
We quantified the resistance levels of transgenic rice plants, expressing Myxococcus xanthus protoporphyrinogen oxidase (PROTOX) in chloroplasts and mitochondria, to PROTOX inhibitors, acifluorfen, oxyfluorfen, carfentrazone-ethyl, and oxadiazon. We also determined whether active oxygen species-scavenging enzymes are involved in the resistance mechanism of transgenic rice. The transgenic rice line M4 was about >200-fold more resistant to oxyfluorfen than the wild-type (WT).
Ha Il Jung; Yong In Kuk; Kyoungwhan Back; Nilda R. Burgos
Comparison of DNA Walking Methods for Isolation of Transgene-Flanking Regions in GM Potato
An important aspect in the safety assessment of transgenic plants is the exact location of transgene insertion sites within\\u000a the host genome. However, robust standard operating procedures are not currently available. Using potato as a test species,\\u000a different methodologies for the determination of insertion sites using a range of published protocols and commercially available\\u000a kits were assessed in transgenic lines
Danny Cullen; Wendy Harwood; Mark Smedley; Howard Davies; Mark Taylor
High-level expression of bovine ?s1-casein in milk of transgenic mice
The bovine as1-casein gene, isolated from a cosmid library, was introduced into the murine germline. Transgene expression occurred in all transgenic mice, and was confined to the lactating mammary gland. Half of the mouse lines (five out of ten) expressed at relatively high expression levels (>1 mg ml-1). The highest levels of expression were obtained with a transgene containing 14.2
MONIQUE Rijnkels; Patricia M. Kooiman; Gerard J. Platenburg; Mieneke van Dixhoorn; Jan H. Nuijens; Herman A. de Boer; Frank R. Pieper
Recloned transgenic pigs possess normal reproductive performance and stable genetic transmission capacity.
The present study investigated whether a recloning procedure would affect the reproductive performance or the germline transmission capacity of recloned transgenic pigs. This study has also laid the foundation for the development of elite transgenic swine breeds in the future. Recloned transgenic pigs were developed from ear tissue fibroblasts of primary transgenic cloned pigs using a recloning procedure, and their reproductive performance and exogenous gene transmission were analyzed. Two transgenic cell lines with different genetic backgrounds (derived from a female miniature pig and a male Landrace pig) with stable expression of green fluorescent protein (GFP) were established successfully. Furthermore, recloned transgenic embryos were developed to full term successfully. One female Chinese experimental miniature piglet (CEMP) (GFP+) and three male Landrace piglets (GFP+) were delivered naturally. Furthermore, the index values for the reproductive characteristics of the recloned transgenic pigs, such as puberty, gestation period, sperm volume and sperm concentration, were not significantly different from those of conventionally bred pigs. In addition, 53% of the F1 offspring of the recloned transgenic pigs were GFP positive. These results demonstrate that ear tissue fibroblasts from primary transgenic cloned pigs efficiently support the full-term development of recloned transgenic embryos. Furthermore, recloned transgenic pigs maintain normal reproductive performance and stable germline (genetic) transmission capacities. PMID:22784554
Cao, Zubing; Li, Yan; Wen, Xiao; Li, Zhiyuan; Mi, Changsheng; Zhang, Zaihu; Li, Ning; Li, Qiuyan
Pharmacogenetic heterogeneity of transgene expression in muscle and tumours
Background Recombinant adenoviruses are employed to deliver a therapeutic transgene in the liver, muscle or tumour tissue. However, to rationalise this delivery approach, the factors of variation between individuals need to be identified. It is assumed that differences between inbred strains of laboratory animals are considered to reflect differences between patients. Previously we showed that transgene expression in the liver of different rat strains was dependent on the transcription efficiency of the transgene. In the present paper we investigated if transfection of muscle and tumour tissue were also subject to such variations. Methods Variation, in transgene expression, after intramuscular gene delivery was determined in different rodent strains and gene expression in tumours was investigated in different human and rodent cell lines as well as in subcutaneously implanted rodent tumours. The molecular mechanisms involved in transgene expression were dissected using an adenovirus encoding luciferase. The luciferase activity, the viral DNA copies and the luciferase transcripts were assessed in cultured cells as well as in the tissues. Results Large differences of luciferase activity, up to 2 logs, were observed between different rodent strains after intramuscular injection of Ad Luciferase. This inter-strain variation of transgene expression was due to a difference in transcription efficiency. The transgene expression level in tumour cell lines of different tissue origin could be explained largely by the difference of infectibility to the adenovirus. In contrast, the main step responsible for luciferase activity variation, between six human breast cancer cell lines with similar phenotype, was at the transcriptional level. Conclusion Difference in transcriptional efficiency in muscles as observed between different inbred strains and between human breast cancer cell lines may be expected to occur between individual patients. This might have important consequences for clinical gene therapy. The variation between tumour types and tissues within a species are mainly at the levels of infectivity.
Lefesvre, Pierre; Attema, Joline; van Bekkum, Dirk
NASA Technical Reports Server (NTRS)
With rapid world population growth and declining availability of fresh water and arable land, a new technology is urgently needed to enhance agricultural productivity. Recent discoveries in the field of crop transgenics clearly demonstrate the great potential of this technology for increasing food production and improving food quality while preserving the environment for future generations. In this review, we briefly discuss some of the recent achievements in crop improvement that have been made using gene transfer technology.
Li, Y.; Wu, Y. H.; McAvoy, R.; Duan, H.
Transgenic control of perforin gene expression
Perforin is a pore-forming effector molecule of CTL and NK cells. To characterize perforin gene expression and its transcriptional control mechanisms in vivo, expression of a cell surface tag, i.e., human CD4, was driven by 5.1 kb of the murin perforin 5{prime} flanking and promoter region in transgenic mice. Six out of seven transgenic lines expressed the perforin-tag hybrid gene at low to intermediate levels, depending on the integration site. Transgene expression occurred in all cells that physiologically are able to express perforin. At the whole organ level, significant amounts of transgenic mRNA and endogenous perforin mRNA were co-expressed in the lymphoid organs, as well as in the lung, the ileum, the oviduct/uterus, and the bone marrow. At the single cell level, the perforin tag was present on NK cells and on CD8{sup +}, as well as on CD4{sup +} cells. Also targeted were Thy-1.2{sup +} {gamma}{delta} T cells, but not Thy-1.2{sup -} {gamma}{delta} T cells, B cells, nor monocytes. During thymic T cell development, transgene expression occurred in double negative (CD4{sup -}CD8{sup -}) thymocytes and was detected at all subsequent stages, but exceeded the expression levels of the endogenous gene in the thymus. In conclusion, the analyzed perforin 5{prime} flanking and promoter region contains important cis-acting sequences that restrict perforin expression to T cells and NK cells, and therefore provides a unique tool for manipulating T cell and/or Nk cell-mediated immune responses in transgenic mice. On the other hand, the normal control of perforin gene expression involves at least one additional negative control mechanism that was not mediated by the transgenic promoter and upstream region. This control restricts perforin gene expression in thymically developing T cells and in most resting peripheral T cells, but can be released upon T cell activation. 43 refs., 7 figs., 1 tab.
Lichtenheld, M.G.; Podack, E.R.; Levy, R.B. [Univ. of Miami, FL (United States)
[Effects of phytase transgenic corn planting on soil nematode community].
A healthy soil ecosystem is essential for nutrient cycling and energy conversion, and the impact of exogenous genes from genetically modified crops had aroused wide concerns. Phytase transgenic corn (i. e., the inbred line BVLA430101) was issued a bio-safety certificate on 27 September 2009 in China, which could improve the efficiency of feed utilization, reduce environmental pollution caused by animal manure. In this study, the abundance of trophic groups, community structure and ecological indices of soil nematodes were studied over the growing cycle of phytase transgenic corn (ab. transgenic corn) and control conventional parental corn (ab. control corn) in the field. Totally 29 and 26 nematode genera were isolated from transgenic corn and control corn fields, respectively. The abundances of bacterivores and omnivores-predators, the total number of soil nematodes, and the Shannon index (H) were significantly greater under transgenic corn than under control corn, while the opposite trend was found for the relative abundance of herbivores and the maturity index (Sigma MI) of soil nematodes. Repeated-measures analysis of variance (ANOVA) did not detect any significant effects of transgenic corn on the composition and abundance of nematode trophic groups and ecological indices of soil nematodes. Furthermore, the Student-T test showed that the abundances of bacterivores and omnivores-predators and the total number of soil nematodes during the milk-ripe stage were significant higher in the transgenic corn field than in the control corn field. The effects of transgenic corn planting on soil nematodes might be related to the increase in the nitrogen content of field soil under transgenic corn compared to control corn. PMID:25011306
Zhao, Zong-Chao; Su, Ying; Mou, Wen-Ya; Liu, Man-Qiang; Chen, Xiao-Yun; Chen, Fa-Jun
High level production of human growth hormone in the milk of transgenic mice: the upstream region of the rabbit whey acidic protein (WAP) gene targets transgene expression to the mammary gland
The 5? flanking region (6.3 kb) of the rabbit WAP (rWAP) gene possesses important regulatory elements. This region was linked to the human growth hormone (hGH) structural gene in order to target transgene expression to the mammary gland. Thirteen lines of transgenic mice were produced. Milk could be collected from six lines of transgenic mice. In five of them, hGH
Eve Devinoy; Dominique Thepot; Marie-Georges Stinnakre; Marie-Louise Fontaine; Henri Grabowski; Claudine Puissant; Andrea Pavirani; Louis-Marie Houdebine
Ectopic over-expression of peroxisomal ascorbate peroxidase (SbpAPX) gene confers salt stress tolerance in transgenic peanut (Arachis hypogaea).
Peroxisomal ascorbate peroxidase gene (SbpAPX) of an extreme halophyte Salicornia brachiata imparts abiotic stress endurance and plays a key role in the protection against oxidative stress. The cloned SbpAPX gene was transformed to local variety of peanut and about 100 transgenic plants were developed using optimized in vitro regeneration and Agrobacterium mediated genetic transformation method. The T0 transgenic plants were confirmed for the gene integration; grown under controlled condition in containment green house facility; seeds were harvested and T1 plants were raised. Transgenic plants (T1) were further confirmed by PCR using gene specific primers and histochemical GUS assay. About 40 transgenic plants (T1) were selected randomly and subjected for salt stress tolerance study. Transgenic plants remained green however non-transgenic plants showed bleaching and yellowish leaves under salt stress conditions. Under stress condition, transgenic plants continued normal growth and completed their life cycle. Transgenic peanut plants exhibited adequate tolerance under salt stress condition and thus could be explored for the cultivation in salt affected areas for the sustainable agriculture. PMID:24954532
Singh, Natwar; Mishra, Avinash; Jha, Bhavanath
Requirements for green fluorescent protein detection in transgenic zebrafish embryos
We have generated transgenic (Tg) lines of zebrafish in which the green fluorescent protein (GFP)-encoding gfp cDNA is driven by the Xenopus laevis ef1 ? enhancer\\/promoter; Tg embryos from most of these lines show detectable fluorescence throughout their body. We have investigated the copy number of the Tg genes in fluorescent and nonfluorescent lines, in order to determine how this
Adam Amsterdam; Shuo Lin; Larry G. Moss; Nancy Hopkins
Tunneling at ?T=1 in quantum Hall bilayers
NASA Astrophysics Data System (ADS)
Interlayer tunneling measurements in the strongly correlated bilayer quantized Hall phase at ?T=1 are reported. The maximum, or critical, current for tunneling at ?T=1 is shown to be a well-defined global property of the coherent phase, insensitive to extrinsic circuit effects and the precise configuration used to measure it, but also exhibiting a surprising scaling behavior with temperature. Comparisons between the experimentally observed tunneling characteristics and a recent theory are favorable at high temperatures, but not at low temperatures where the tunneling closely resembles the dc Josephson effect. The zero-bias tunneling resistance becomes extremely small at low temperatures, vastly less than that observed at zero magnetic field, but nonetheless remains finite. The temperature dependence of this tunneling resistance is similar to that of the ordinary in-plane resistivity of the quantum Hall phase.
Nandi, D.; Khaire, T.; Finck, A. D. K.; Eisenstein, J. P.; Pfeiffer, L. N.; West, K. W.
Recent Results of IRAN-T1 Tokamak
In this article after introducing the IR-T1 tokamak and its diagnostic systems a brief discussion on the range of grossly stable operating conditions of its plasma by Hugill diagram is presented. Hard disruption instability is studied experimentally in the next part, which confirms that MHD behavior in small tokamaks can be characterized by a single parameter q(a), safety factor at plasma edge. Finally the characteristics of the new regime of IR-T1 are reported. By our new model of triggering different fields (toroidal, ohmic and vertical), the plasma duration time is increased up to 35 ms with Ip of about 25 kA. By modifying capacitance and charging voltage of ohmic and vertical fields the spike oscillations which was appeared in the plasma behavior is taken out. The role of cleaning the vacuum chamber and using heavier gas for glow discharge and the effect of base pressure is described in detail.
Dorranian, D.; Ghoranneviss, M.; Salem, M. K.; Mahmoodi D, M.; Arvin, R.; Talebitaher, Alireza; Abhari, Ali [Plasma Physics Research Center, Science and Research Campus, Islamic Azad University, Poonak, Hesarak, Tehran (14736) (Iran, Islamic Republic of); Khorshid, P. [Physics Dept., Mashhad Campus, Islamic Azad University, Mashhad (Iran, Islamic Republic of); Hojabri, A. [Physics Dept., Karaj Campus, Islamic Azad University, Karaj (Iran, Islamic Republic of)
Trends in surgical management of T1 renal cell carcinoma.
Historically, open radical nephrectomy (ORN) represented the standard of care for localized renal cell carcinoma (RCC). While the incidence of T1 RCC is rising, treatment options are developing fast and the standard of care according to European and American guidelines has changed to partial nephrectomy (PN), or laparoscopic radical nephrectomy in patients not suitable for PN. To assess the implementation of guideline recommendations and to profile recent surgical and technical innovations, we reviewed the current literature. We observed that ORN still represents the most commonly used treatment in T1 RCC patients. Utilization of PN increased over time but implementation is still in progress. Whereas PN is frequently used in tertiary care centers, population-based studies suggest discrepancies in the diffusion of standard of care treatments. Alternative minimally invasive approaches for PN are available but their superiority is not yet proven. Further efforts in improving the training of urologic surgeons are required to continue the implementation of guideline recommendations. PMID:24414526
Schiffmann, Jonas; Bianchi, Marco; Sun, Maxine; Becker, Andreas
Tandem constructs to mitigate transgene persistence: tobacco as a model.
Some transgenic crops can introgress genes into other varieties of the crop, to related weeds or themselves remain as 'volunteer' weeds, potentially enhancing the invasiveness or weediness of the resulting offspring. The presently suggested mechanisms for transgene containment allow low frequency of gene release (leakage), requiring the mitigation of continued spread. Transgenic mitigation (TM), where a desired primary gene is tandemly coupled with mitigating genes that are positive or neutral to the crop but deleterious to hybrids and their progeny, was tested as a mechanism to mitigate transgene introgression. Dwarfism, which typically increases crop yield while decreasing the ability to compete, was used as a mitigator. A construct of a dominant ahasR (acetohydroxy acid synthase) gene conferring herbicide resistance in tandem with the semidominant mitigator dwarfing Delta gai (gibberellic acid-insensitive) gene was transformed into tobacco (Nicotiana tabacum). The integration and the phenotypic stability of the tandemly linked ahasR and Delta gai genomic inserts in later generations were confirmed by polymerase chain reaction. The hemizygous semidwarf imazapyr-resistant TM T1 (= BC1) transgenic plants were weak competitors when cocultivated with wild type segregants under greenhouse conditions and without using the herbicide. The competition was most intense at close spacings typical of weed offspring. Most dwarf plants interspersed with wild type died at 1-cm, > 70% at 2.5-cm and 45% at 5-cm spacing, and the dwarf survivors formed no flowers. At 10-cm spacing, where few TM plants died, only those TM plants growing at the periphery of the large cultivation containers formed flowers, after the wild type plants terminated growth. The highest reproductive TM fitness relative to the wild type was 17%. The results demonstrate the suppression of crop-weed hybrids when competing with wild type weeds, or such crops as volunteer weeds, in seasons when the selector (herbicide) is not used. The linked unfitness would be continuously manifested in future generations, keeping the transgene at a low frequency. PMID:14871372
Al-Ahmad, Hani; Galili, Shmuel; Gressel, Jonathan
Molecular structures in T=1 states of 10B
NASA Astrophysics Data System (ADS)
Background: Multicenter (molecular) structures can play an important role in light nuclei. The highly deformed rotational band in 10Be with a bandhead at 6.179 MeV has been observed recently and suggested to have an exotic ?:2n:? configuration.Purpose: Search for states with ?:pn:? two-center molecular configurations in 10B that are analogous to the states with ?:2n:? structure in 10Be.Methods: The T=1 isobaric analog states in 10B were studied in the energy range of Ex=8.7--12.1 MeV using the reaction 1H(9Be,?)6Li*(T=1, 0+, 3.56 MeV). An R-matrix analysis was used to extract parameters for the states observed in the (p,?) excitation function.Results: Five T=1 states in 10B have been identified. The known 2+ and 3- states at 8.9 MeV have been observed and their partial widths have been measured. The spin parities and partial widths for three higher-lying states were determined.Conclusions: Our data support theoretical predictions that the 2+ state at 8.9 MeV (isobaric analog of the 7.54-MeV state in 10Be) is a highly clustered state and can be identified as a member of the ?:np:? rotational band. The next member of this band, the 4+ state, has not been found. A very broad 0+ state at 11 MeV that corresponds to pure ?+6Li(0+, T=1) configuration is suggested and it might be related to similar structures found in 12C, 18O, and 20Ne.
Kuchera, A. N.; Rogachev, G. V.; Goldberg, V. Z.; Johnson, E. D.; Cherubini, S.; Gulino, M.; La Cognata, M.; Lamia, L.; Romano, S.; Miller, L. E.; Pizzone, R. G.; Rapisarda, G. G.; Sergi, M. L.; Spitaleri, C.; Tribble, R. E.; Trzaska, W. H.; Tumino, A.
Recent Results of IRAN-T1 Tokamak
In this article after introducing the IR-T1 tokamak and its diagnostic systems a brief discussion on the range of grossly stable operating conditions of its plasma by Hugill diagram is presented. Hard disruption instability is studied experimentally in the next part, which confirms that MHD behavior in small tokamaks can be characterized by a single parameter q(a), safety factor at
D. Dorranian; M. Ghoranneviss; M. K. Salem; M. Mahmoodi D; R. Arvin; Alireza Talebitaher; Ali Abhari; P. Khorshid; A. Hojabri
Mechanism of Al2CuLi ( T 1) nucleation and growth
NASA Astrophysics Data System (ADS)
Conventional strain contrast transmission electron microscopy (CTEM) and high-resolution transmission electron microscopy (HRTEM) were performed to establish the nucleation and growth mechanism of Al2CuLi (T1) precipitates in an Al-Li-Cu alloy. It is shown that the growth mechanism of T 1 precipitate plates occurs by the diffusional glide of growth ledges composed of b = 1/6<112> partial dislocations on 111 matrix planes and that the growth ledges migrate by the ledge-kink mechanism, as previously suggested by Cassada et al. 1 for this system. T 1 plate nucleation is modeled as the dissociation of a perfect b = 1/2<110> matrix dislocation in the vicinity of a dislocation jog. The coordinated dissociation of the dislocation line segments on each side of the sessile jog provides the displacement necessary for the formation of a new hexagonal plate or plate ledge. Strain contrast analysis of the Burgers vector of plate edges and the edges of growth ledges indicates the stacking of partial dislocations is of mixed displacement. Formerly Graduate Student, Department of Materials Science, University of Virginia,
Cassada, W. A.; Shiflet, G. J.; Starke, E. A.
Computerized study with 201T1 of the cold thyroid node.
Because of its physical and potassium-metabolic characteristics 201T1 is more suitable than 131Cs for radioisotopic studies of the cold thyroid nodule, with the further diagnostic possibility of quantitatively assessing intranodular behavior for a specific differentiation among different kinds of neoformations. Using a gamma-camera on line with a computer data processing device, sequential scintiscans were recorded for the first 20-30 min after i.v. administration of 15-20 microCi/kg of radiothallium; delayed sequences were taken at 40-60 min if intranodular uptake appeared. A quantitative appraisal was made of the differential 201T1 uptake-ratio between nodule and healthy thyroid tissue (density-index) and the multiparameter analysis of thyroid time/activity curves generated on the relative regions of interest (ROIs). This computerized study, in 120 out of 293 patients submitted to this radiothallium test, has shown a) diagnostic agreement between clinical-histological and radioisotopic findings in 76 out of 79 colloid-cystic or degenerative neoformations, in all 16 malignant and in 23 out of 25 hyperplastic benign nodules; b) significant statistical difference of the density-index in solid versus cystic but not between benign and malignant nodules; c) different 201T1 kinetics behaviour in different kinds of solid thyroid lesions with a satisfactory statistical difference of the radiothallium nodular disappearance-index. PMID:6281742
Palermo, F; Saitta, B; Coghetto, F; Tiberio, M; Caldato, L
Microinjection of cre recombinase RNA induces site-specific recombination of a transgene in mouse oocytes.
We have developed a strategy for producing single copy transgenic mouse lines using Cre-loxP site specific recombination. The method is based on transient expression of the recombinase after injection of in vitro transcribed mRNA into the cytoplasm of fertilised eggs containing multiple copies of the transgene. The success rate of the recombination event is 100% (15 out of 15).
de Wit, T; Drabek, D; Grosveld, F
Novel Hippocampal Interneuronal Subtypes Identified Using Transgenic Mice That Express Green Fluorescent Protein in GABAergic Interneurons
The chief inhibitory neurons of the mammalian brain, GABAer- gic neurons, are comprised of a myriad of diverse neuronal subtypes. To facilitate the study of these neurons, transgenic mice were generated that express enhanced green fluorescent protein (EGFP) in subpopulations of GABAergic neurons. In one of the resulting transgenic lines, called GIN (GFP-expressing Inhibitory Neurons), EGFP was found to be
Anthony A. Oliva Jr; Minghui Jiang; Trang Lam; Karen L. Smith; John W. Swann
Targeted expression of a conditional oncogene in hematopoietic cells of transgenic mice
We have produced two lines of transgenic mice in which the expression of temperature-sensitive SV-40 large T antigen is targeted to bone marrow megakaryocytes via the platelet factor 4 (PF4) tissue- specific promoter. The progeny of these transgenic mice were observed for about 3 mo, and no malignan- cies were detected over this period of time. The offspring of these
Katya Ravid; Yah Chun Li; Helen B. Rayburn; Robert D. Rosenberg
Imaging Neuronal Subsets in Transgenic Mice Expressing Multiple Spectral Variants of GFP
We generated transgenic mice in which red, green, yellow, or cyan fluorescent proteins (together termed XFPs) were selectively expressed in neurons. All four XFPs labeled neurons in their entirety, including axons, nerve terminals, dendrites, and dendritic spines. Remarkably, each of 25 independently generated transgenic lines expressed XFP in a unique pattern, even though all incorporated identical regulatory elements (from the
Guoping Feng; Rebecca H. Mellor; Michael Bernstein; Cynthia Keller-Peck; Quyen T. Nguyen; Mia Wallace; Jeanne M. Nerbonne; Jeff W. Lichtman; Joshua R. Sanes
Two Amyloid Precursor Protein Transgenic Mouse Models with Alzheimer Disease-Like Pathology
Mutations in the amyloid precursor protein (APP) gene cause early-onset familial Alzheimer disease (AD) by affecting the formation of the amyloid beta (Abeta ) peptide, the major constituent of AD plaques. We expressed human APP751 containing these mutations in the brains of transgenic mice. Two transgenic mouse lines develop pathological features reminiscent of AD. The degree of pathology depends on
Christine Sturchler-Pierrat; Dorothee Abramowski; Mairead Duke; Karl-Heinz Wiederhold; Claudia Mistl; Sabin Rothacher; Brigit Ledermann; Kurt Burki; Peter Frey; Paolo A. Paganetti; Caroline Waridel; Michael E. Calhoun; Mathias Jucker; Alphonse Probst; Matthias Staufenbiel; Bernd Sommer
Dramatically accelerated growth and extraordinary gigantism of transgenic mud loach Misgurnus mizolepis.
Transgenic mud loaches (Misgurnus mizolepis), in which the entire transgene originated from the same species, have been generated by microinjecting the mud loach growth hormone (mlGH) gene fused to the mud loach beta-actin promoter. Out of 4,100 eggs injected, 7.5% fish derived from the injected eggs showed dramatically accelerated growth, with a maximum of 35-fold faster growth than their non-transgenic siblings. Many fast-growing transgenic individuals showed extraordinary gigantism: their body weight and total length (largest fish attained to 413 g and 41.5 cm) were larger and longer than even those of 12-year-old normal broodstock (maximum size reached to 89 g and 28 cm). Of 46 transgenic founders tested, 30 individuals transmitted the transgene to next generation with a wide range of germ-line transmission frequencies ranging from 2% to 33%. The growth performance of the subsequent generation (F1) was also dramatically accelerated up to 35-fold, although the levels of enhanced growth were variable among transgenic lines. Three transgenic germ-lines up to F4 were established, showing the expected Mendelian inheritance of the transgene. Expression of GH mRNA in many tissues was detected by RT-PCR analyses. The time required to attain marketable size (10 g) in these transgenic lines was only 30-50 days after fertilization, while at least 6 months in non-transgenic fish. Besides growth enhancement, significantly improved feed-conversion efficiency up to 1.9-fold was also observed. PMID:11592714
Nam, Y K; Noh, J K; Cho, Y S; Cho, H J; Cho, K N; Kim, C G; Kim, D S
Identification, cloning and characterization of a plasma membrane zinc efflux transporter, TrZnT-1, from fugu pufferfish (Takifugu rubripes)
An orthologue of the mammalian ZnT-1 (zinc transporter-1) gene was cloned from the intestine of the torafugu pufferfish (Takifugu rubripes), demonstrating that this gene predates the evolution of land-living vertebrates. TrZnT-1 (T. rubripes ZnT-1) shares overall topology with other members of the ZnT-1 family of zinc transporters, with six TMs (transmembrane domains) including a large histidine-rich intracellular loop between TM IV and V and intracellular C- and N-termini. Expression of TrZnT-1 in a metallothionein acquiescent cell line suggested that this protein reduces intracellular Zn2+ levels. Manipulation of the transporting media showed that several externally applied hydrominerals had no effect on TrZnT-1 activity. However, addition of N-ethylmaleimide increased TrZnT-1-mediated transport, possibly by increasing intracellular free Zn2+ levels by Zn2+ release from carrier proteins. Generation of a specific antibody and subsequent immunocytochemistry on fixed cells overexpressing TrZnT-1 indicated that the protein is localized to the plasma membrane in these cells. The genomic organization of TrZnT-1 is the same as that in mammals with two exons. The upstream regulatory region of the TrZnT-1 gene contains several putative cis-acting elements, including metal-response elements and an Sp1 site. Analysis of the DNA contigs surrounding the TrZnT-1 gene reveal limited synteny between corresponding regions in the rat, mouse and human; however, this was very low, with only two syntenic genes, ZnT-1 and NEK2 (never in mitosis gene A-related kinase).
Balesaria, Sara; Hogstrand, Christer
Comprehensive Assessment of Milk Composition in Transgenic Cloned Cattle
The development of transgenic cloned animals offers new opportunities for agriculture, biomedicine and environmental science. Expressing recombinant proteins in dairy animals to alter their milk composition is considered beneficial for human health. However, relatively little is known about the expression profile of the proteins in milk derived from transgenic cloned animals. In this study, we compared the proteome and nutrient composition of the colostrum and mature milk from three lines of transgenic cloned (TC) cattle that specifically express human ?-lactalbumin (TC-LA), lactoferrin (TC-LF) or lysozyme (TC-LZ) in the mammary gland with those from cloned non-transgenic (C) and conventionally bred normal animals (N). Protein expression profile identification was performed, 37 proteins were specifically expressed in the TC animals and 70 protein spots that were classified as 22 proteins with significantly altered expression levels in the TC and C groups compared to N group. Assessment of the relationship of the transgene effect and normal variability in the milk protein profiles in each group indicated that the variation in the endogenous protein profiles of the three TC groups was within the limit of natural variability. More than 50 parameters for the colostrum and mature milk were compared between each TC group and the N controls. The data revealed essentially similar profiles for all groups. This comprehensive study demonstrated that in TC cattle the mean values for the measured milk parameters were all within the normal range, suggesting that the expression of a transgene does not affect the composition of milk.
Sui, Shunchao; Yu, Tian; Wang, Jianwu; Li, Ning
Neurologic and motor dysfunctions in APP transgenic mice
The discovery of gene mutations underlying autosomal dominant AlzheimerÆs disease has enabled researchers to reproduce several hallmarks of this disorder in transgenic mice, notably the formation of A? plaques in brain and cognitive deficits. APP transgenic mutants have also been investigated with respect to survival rates, neurologic functions, and motor coordination, which are all susceptible to alteration in Alzheimer dementia. Several transgenic lines expressing human mutated or wild-type APP had higher mortality rates than non-transgenic controls with or without the presence of A? plaques. Mortality rates were also elevated in APP transgenic mice with vascular amyloid accumulation, thereby implicating cerebrovascular factors in the precocious death observed in all APP transgenic models. In addition, myoclonic jumping has been described in APP mutants, together with seizure activity, abnormal limb-flexion and paw-clasping reflexes, and motor coordination deficits. The neurologic signs resemble the myoclonic movements, epileptic seizures, pathological reflexes, and gait problems observed in late-stage AlzheimerÆs disease.
Lalonde, Robert; Fukuchi, Ken-ichiro; Strazielle, Catherine
Insect-resistant transgenic plants
The technology of insect-resistant transgenic plants is expanding very rapidly, with considerable research activity in both the private and public sectors. The only commercialized insect-resistant transgenic plants to date express genes derived from the bacterium Bacillus thuringiensis, but a wide range of genes from higher plants have also been transferred into crop cultivars, especially genes encoding inhibitors of digestive enzymes
Tanja H Schuler; Guy M Poppy; Brian R Kerry; Ian Denholm
Recombineering strategies for developing next generation BAC transgenic tools for optogenetics and beyond
The development and application of diverse BAC transgenic rodent lines has enabled rapid progress for precise molecular targeting of genetically-defined cell types in the mammalian central nervous system. These transgenic tools have played a central role in the optogenetic revolution in neuroscience. Indeed, an overwhelming proportion of studies in this field have made use of BAC transgenic Cre driver lines to achieve targeted expression of optogenetic probes in the brain. In addition, several BAC transgenic mouse lines have been established for direct cell-type specific expression of Channelrhodopsin-2 (ChR2). While the benefits of these new tools largely outweigh any accompanying challenges, many available BAC transgenic lines may suffer from confounds due in part to increased gene dosage of one or more ōextraö genes contained within the large BAC DNA sequences. Here we discuss this under-appreciated issue and propose strategies for developing the next generation of BAC transgenic lines that are devoid of extra genes. Furthermore, we provide evidence that these strategies are simple, reproducible, and do not disrupt the intended cell-type specific transgene expression patterns for several distinct BAC clones. These strategies may be widely implemented for improved BAC transgenesis across diverse disciplines.
Ting, Jonathan T.; Feng, Guoping
A 90-Day Dietary Toxicity Study of Genetically Modified Rice T1C-1 Expressing Cry1C Protein in Sprague Dawley Rats
In a 90-day study, Sprague Dawley rats were fed transgenic T1C-1 rice expressing Cry1C protein and were compared with rats fed non-transgenic parental rice Minghui 63 and rats fed a basal diet. No adverse effects on animal behavior or weight gain were observed during the study. Blood samples were collected and analyzed, and standard hematological and biochemical parameters were compared. A few of these parameters were found to be significantly different, but were within the normal reference intervals for rats of this breed and age, and were thus not considered to be treatment-related. Following sacrifice, a large number of organs were weighed, and macroscopic and histopathological examinations were performed with no changes reported. The aim of this study was to use a known animal model to determine the safety of the genetically modified (GM) rice T1C-1. The results showed no adverse or toxic effects due to T1C-1 rice when tested in this 90-day study.
Tang, Xueming; Han, Fangting; Zhao, Kai; Xu, Yan; Wu, Xiao; Wang, Jinbin; Jiang, Lingxi; Shi, Wei
[Enhanced biosynthesis of scopolamine in transgenic Atropa belladonna by overexpression of h6h gene].
Transgenic Atropa belladonna with high levels of scopolamine was developed by metabolic engineering. A functional gene involved in the rate limiting enzyme of h6h involved in the biosynthetic pathway of scopolamine was over expressed in A. belladonna via Agrobacterium-mediation. The transgenic plants were culturing till fruiting through micropropogating and acclimating. The integration of the h6h genes into the genomic DNA of transgenic plants were confirmed by genomic polymerase chain reaction (PCR) analysis. Analysis of the difference of plant height, crown width, stem diameter, leaf length, leaf width, branch number and fresh weight was carried out using SPSS software. The content of hyoscyamine and scopolamine in roots, stems, leaves and fruits was determined by HPLC. The investigation of the expression levels of Hnh6h by qPCR. Both Kan(r) and Hnh6h genes were detected in five transgenic lines of A. belladonna plants (A8, A11, A12, C8 and C19), but were not detected in the controls. The plant height, crown width, stem diameter, leaf length, leaf width, branch number and fresh weight of transgenic plants did not decrease by comparison with the non-transgenic ones, and furthermore some agronomic characters of transgenic plants were better than those of the controls. The highest level of scopolamine was found in leaves of transgenic A. belladonna, and the content of scopolamine was also higher than that of hyoscyamine in leaves. The contents of scopolamine of leaves in different transgenic lines were listed in order: C8 > A12 > C19 > A11 > A8, especially, the content of scopolamine in transgenic line C8 was 2.17 mg x g(-1) DW that was 4.2 folds of the non-transgenic ones (0.42 mg x g(-1) DW). The expression of transgenic Hnh6h was detected in all the transgenic plants but not in the control. The highest level of Hnh6h expression was found in transgenic leaves. Overexpression of Hnh6h is able to break the rate limiting steps involved in the downstream pathway of scopolamine biosynthesis, and thus promotes the metabolic flux flowing toward biosynthesis of scopolamine to improve the capacity of scopolamine biosynthesis in transgenic plants. As a result, transgenic plants of A. belladonna with higher level of scopolamine were developed. PMID:24010284
Li, Jin-Di; Qin, Bai-Fu; Yang, Chun-Xian; Lan, Xiao-Zhong; Wu, Neng-Biao; Liao, Zhi-Hua
Exercise and type 1 diabetes (T1DM).
Physical exercise is firmly incorporated in the management of type 1 diabetes (T1DM), due to multiple recognized beneficial health effects (cardiovascular disease prevention being preeminent). When glycemic values are not excessively low or high at the time of exercise, few absolute contraindications exist; practical guidelines regarding amount, type, and duration of age-appropriate exercise are regularly updated by entities such as the American Diabetes Association and the International Society for Pediatric and Adolescent Diabetes. Practical implementation of exercise regimens, however, may at times be problematic. In the poorly controlled patient, specific structural changes may occur within skeletal muscle fiber, which is considered by some to be a disease-specific myopathy. Further, even in well-controlled patients, several homeostatic mechanisms regulating carbohydrate metabolism often become impaired, causing hypo- or hyperglycemia during and/or after exercise. Some altered responses may be related to inappropriate exogenous insulin administration, but are often also partly caused by the "metabolic memory" of prior glycemic events. In this context, prior hyperglycemia correlates with increased inflammatory and oxidative stress responses, possibly modulating key exercise-associated cardio-protective pathways. Similarly, prior hypoglycemia correlates with impaired glucose counterregulation, resulting in greater likelihood of further hypoglycemia to develop. Additional exercise responses that may be altered in T1DM include growth factor release, which may be especially important in children and adolescents. These multiple alterations in the exercise response should not discourage physical activity in patients with T1DM, but rather should stimulate the quest for the identification of the exercise formats that maximize beneficial health effects. PMID:23897688
Galassetti, Pietro; Riddell, Michael C
The TOTEM T1 read out card motherboard
NASA Astrophysics Data System (ADS)
This article describes the Read Out Card (ROC) motherboard, which is the main component of the T1 forward telescope front-end electronic system. The ROC main objectives are to acquire tracking data and trigger information from the detector. It performs data conversion from electrical to optical format and transfers the data streams to the next level of the system and it implements Slow Control modules which are able to receive, decode and distribute the LHC machine low jitter clock and fast command. The ROC also provides a spy mezzanine connection based on programmable FPGA and USB2.0 for laboratory and portable DAQ debugging system.
Minutoli, S.; Lo Vetere, M.; Robutti, E.
A transgenic mouse model for lung adenocarcinoma.
Lung cancer is a leading cause of tumor-related deaths in humans but its origin and development are poorly understood. To study the biology of these tumors, appropriate animal and cell culture models will be of eminent importance. Uteroglobin is a marker protein for the nonciliated epithelial Clara cells lining the respiratory and terminal bronchioli of the lung. We have used the promoter and 5'-flanking sequences of the rabbit uteroglobin gene to target expression of the SV40 T antigen to the lung of transgenic mice. All transgenic founders as well as the descendants from an established line, UT7.1, developed multifocal bronchioloalveolar adenocarcinomas originating from Clara cells. At least three different stages in tumor development with progressive loss of the differentiated phenotype can be distinguished by immunohistochemical data and in situ hybridization. Only in the initial stage did bronchiolar cells express both uteroglobin and SV40 T antigen, whereas at later stages, only SV40 T antigen was detected, and the most advanced tumors were negative for both proteins. Starting from the lungs of UT7.1 mice, a bronchiolar cell line was established that maintains the features of differentiated Clara cells. This system provides a useful model for further studying the development and progression of lung adenocarcinomas in vivo and in vitro. PMID:7718490
Sandm÷ller, A; Halter, R; Suske, G; Paul, D; Beato, M
Increased resistance to fungal wilts in transgenic eggplant expressing alfalfa glucanase gene.
The wilt diseases caused by Verticillium dahliae and Fusarium oxysporum are the major diseases of eggplant (Solanum melongena L.). In order to generate transgenic resistance against the wilt diseases, Agrobacterium-mediated gene transfer was performed to introduce alfalfa glucanase gene encoding an acidic glucanase into eggplant using neomycin phosphotransferase (npt-II) gene as a plant selection marker. The transgene integration into eggplant genome was confirmed by Polymerase chain reaction (PCR) and Southern blot analysis and transgene expression by the glucanase activity and western blot analysis. The selected transgenic lines were challenged with V. dahliae and F. oxysporum under in vitro and in vivo growth conditions, and transgenic lines showed enhanced resistance against the wilt-causing fungi with a delay of 5-7Ādays in the disease development as compared to wild-type plants. PMID:24757318
Singh, Deepali; Ambroise, Annick; Haicour, Robert; Sihachakr, Darasinh; Rajam, Manchikatla Venkat
Transgenic tobacco plants expressing the maize Cat2 gene have altered catalase levels that affect plant-pathogen interactions and resistance to oxidative stress
Transgenic tobacco genotypes expressing the maize Cat2 gene were developed with altered catalase (CAT) levels that resulted in a moderate increase of CAT activity in two transgenic lines. Bacterial infection, with a pathogen that does not share homology with the transgene, caused local and systemic down-regulation of the steady state mRNA levels of the 35S-driven transgene in a manner resembling
A. N. Polidoros; P. V. Mylona; J. G. Scandalios
[Triptolide inhibits cell proliferation by downregulating phosphorylation of estrogen reporters in 4T1 tumor-bearing mice].
In order to investigate the anti-proliferative effects of triptolide (TP) on 4T1 mice breast cancer cell line in vitro and in mouse model, as well as the possible mechanisms, we detected the effect of TP on cell proliferation by MTT assay or Crystal Violet Staining in our research. Flowcytometry combined with FITC-Annexin V/PI staining were used for detecting TP induced 4T1 cell apoptosis. The protein expression of ERalpha, p-ERalpha, ERbeta, p-ERbeta, ERK, p-ERK, p38, p-p38, SAPK/JNK, and p-SAPK/JNK was tested by western blotting. We also compare TP with chemotherapy drug doxorubicin in 4T1 tumor bearing BLAB/c mice model, the Xenogen bioluminescence imaging, H&E, and IHC result indicated that TP exhibits an anticancer proliferation activity. As a result, TP in 100, 10, 1, 0.1 micromol x L(-1), all inhibited the proliferation of 4T1 cells by MTT assay and Crystal Violet Staining. TP which concentrations is 10, 1, 0.1 micromol x L(-1) could induce the apoptosis of 4T1 cells and reduce the cell proliferation. TP in 200 microg x kg(-1) could inhibit the tumor growth in vivo. The anticancer proliferation of TP was involved in its effect on reducing expression of ERalpha, p-ERalpha, ERbeta, and p-ERbeta, but nothing to do with the activation of MAPK signaling pathway. PMID:24791503
Pan, Guo-Feng; Gao, Jian-Li; Zhang, Qi; Lv, Gui-Yuan; Chen, Su-Hong
Targeting of glutamine synthetase to the mitochondria of transgenic tabacco
Two transgenic tobacco lines were genetically engineered to contain chimaeric genes encoding the glutamine synthetase (GS) ? polypeptide of Phaseolus vulgaris (French bean), expressed from the cauliflower mosaic virus 35S promoter. One (MIT-1) contained two copies of a construct including the first 60 amino acids of the Nicotiana plumbaginifolia ▀-F1 ATPase to target the GS polypeptide to the mitochondrion. The
Pascale Hemon; Mark P. Robbins; Julie V. Cullimore
Identification of secretory odontoblasts using DMP1GFP transgenic mice
Terminal differentiation of odontoblasts from dental papilla is a long process involving several intermediate steps and changes in the transcriptional profile and expression of proteins secreted by cells in the odontoblast lineage. Transgenic mouse lines in which GFP expression is under the control of tissue- and stage specific promoters have provided powerful experimental tools for identification and isolation of cells
Anamaria Balic; Mina Mina
Stable transmission and transcription of newfoundland ocean pout type III fish antifreeze protein (AFP) gene in transgenic mice and hypothermic storage of transgenic ovary and testis.
Here we describe the generation of transgenic mice carrying type III fish antifreeze protein (AFP) gene and evaluate whether AFP type III protects transgenic mouse ovaries and testes from hypothermic storage. AFPs exist in many different organisms. In fish, AFPs protect the host from freezing at temperatures below the colligative freezing point by adsorbing to the surface of nucleating ice crystals and inhibiting their growth. The transgenic expression of AFP holds great promise for conferring freeze-resistant plant and animal species. AFP also exhibits a potential for the cryopreservation of tissues and cells. In this study, we have generated 42 founder mice harboring the Newfoundland ocean pout (OP5A) type III AFP transgene and established one transgenic line (the line #6). This study demonstrated that AFP gene construct has been stably transmitted to the mouse progeny in the F3 generations in the line #6. Furthermore, the presence of AFP transcripts was confirmed by RT-PCR analysis on cDNAs from liver, kidney, ovarian, and testis tissues of the mouse from F3 generation in this line. These results indicate that ocean pout type III AFP gene could be integrated and transmitted to the next generation and stably transcribed in transgenic mice. In histological analysis of testis and ovarian tissues of nontransgenic control and AFP transgenic mice it has been shown that both tissues of AFP transgenic mice were protected from hypothermic storage (+4 degrees C). The AFP III transgenic mice obtained for the first time in this study would be useful for investigating the biological functions of AFP in mammalian systems and also its potential role in cryopreservation. PMID:16894545
Bagis, Haydar; Aktoprakligil, Digdem; Mercan, Hande Odaman; Yurdusev, Nevzat; Turgut, Gazi; Sekmen, Sakir; Arat, Sezen; Cetin, Seyfettin
The Natural History of pT1 Colorectal Cancer
Colorectal carcinoma invading the submucosa but not the muscular layer (pT1, early invasive cancer) represents the earliest form of clinically relevant colorectal cancer in most patients. Neoplastic invasion of the submucosa, in fact, opens the way to metastasis via the lymphatic and blood vessels, and the choice between surveillance and major surgery will turn on its metastatic potential. The following histological features predict the risk of metastasis and the different clinical outcomes: grade of differentiation of carcinoma, lymphovascular invasion, state of the resection margin. Microstaging of invasive cancer, namely the width and the depth of submucosal invasion, together with tumor budding at the advancing edge allow the metastatic risk to be further stratified in minimal, low, and high. Different, although morphologically undistinguishable, tumorigenic pathways are supposed to lead to the malignant transformation of colonic mucosa and subsequently to drive the progression from early to advanced cancer: new biomarkers are needed to identify progressive and non-progressive pT1 neoplasia.
Risio, Mauro
Bacteriophage T1 was suspended in distilled water and in phosphate buffer, saturated with oxygen, nitrogen, hydrogen, and carbon monoxide, and irradiated with gamma rays and x-rays. Under the same conditions phage was exposed to hydrogen peroxide. Oxygen acted as a protective agent against both irradiation and hydrogen peroxide inactivation. As a protective agent against irradiation, oxygen was more efficient in distilled water than in buffer. The phage was much more sensitive to irradiation in the presence of hydrogen or nitrogen than in the presence of oxygen. Survivals of phage irradiated in suspensions saturated with hydrogen and with nitrogen did not differ significantly. From this it was concluded that oxygen did not protect T1 by removing atomic hydrogen from the irradiated medium, since the hydrogen-saturated medium increased the yield of atomic hydrogen but did not increase the yield of inactivated phage. It was presumed, therefore, that phage is sensitive to OH radicals and this was confirmed by irradiating phage with UV in the presence of hydrogen peroxide and comparing this survival with the survivals obtained from hydrogen peroxide alone and from UV alone. The combined effect of hydrogen peroxide and UV acting simultaneously was greater than the effect attributable to hydrogen peroxide and UV acting separately. Evidence for sensitivity to HO2 radicals was considered, and the effect was attributed chiefly to an oxidizing action since phage sensitivity is greater at higher hydrogen ion concentrations, which favor oxidation by HO2 radicals. Since the OH radical is a more efficient oxidizing agent than O-, the former being favored in an acid medium, the latter in an alkaline medium, and since the phage is more sensitive in the first situation than in the second, the present tests proved the importance of oxidation as the mechanism of inactivation. Since some inactivation was encountered when phage was exposed to reducing agents, independently of irradiation, it was concluded that phage is somewhat sensitive to reducing agents, but the inactivation attributable to ionizing radiations is due chiefly to oxidation, against which these reducing agents are very efficient protectors. Under no circumstances did hydrogen peroxide protect T1, whether produced by irradiation in the medium or added beforehand to the medium to be irradiated. The first point was investigated by irradiating T1 in the presence of hydrogen and oxygen combined; this produced a higher yield of hydrogen peroxide but a lower survival of T1. In all these tests phage survival under irradiation was directly correlated with oxygen content of the medium rather than with production of hydrogen peroxide. It is proposed that the protective effect of oxygen is due to a reaction between the phage and oxygen, and this complex confers stability upon the phage.
Bachofer, C. S.; Pottinger, M. Aelred
Integration, expression and inheritance of transgenes in hexaploid oat (Avena sativa L.).
Two oat varieties, Melys (spring variety) and Bulwark (winter variety) were transformed by particle bombardment of primary embryogenic callus using either a ubi-bar-ubi-gus co-integration vector or co-transformed (Melys) with a ubi-bar plasmid together with one of three plasmids containing the beta-glucuronidase (gus) gene under the control of either a rice actin promoter, a CaMV35S promoter or a wheat high molecular weight glutenin promoter. Morphologically normal and fertile transgenic plants were regenerated following callus selection with glufosinate ammonium. Evidence for the integration and functioning of the selectable (bar) and reporter (gus) genes in T0 and T1 plants was confirmed by PCR, Southern hybridisation, fluorescence in situ hybridisation (FISH), histochemical assays, and by progeny analysis. Transformation rates varied from 0.2 to 5.0 lines/plate of callus bombarded, with co-transformation frequencies of 83 to 100%, and co-expression frequencies of 60 to 100%. Copy numbers for the bar and gus gene varied from 3 to 17 and from 2 to 20 respectively. Cell and tissue specific expression of the gus gene was evident from the different promoters, with the HMW glutenin promoter showing endosperm specific expression in T1 seed. No expression of the gus gene under the CaMV35S promoter was detected in any tissues. Progeny analysis provided evidence of Mendelian inheritance of the introduced genes suggesting either one or two unlinked integration sites. This was confirmed by fluorescence in situ hybridisation to chromosome spread preparations. No segregation of the gus gene from the bar gene was observed in any of the progeny derived from co-transformation. PMID:12964869
Perret, Sophie J; Valentine, John; Leggett, J Mike; Morris, Phillip
Effects of repeated cultivation of transgenic Bt cotton on functional bacterial populations in rhizosphere soil
The impact of multiple-year (0-5 years) culti- vation of transgenic Bacillus thuringiensis (Bt) cotton on the functional bacterial populations in rhizosphere soil was investigated. The transgenic Bt ? CpTI cotton line SGK321 and a non-Bt cotton line Shiyuan321 were planted in four fields in which Bt cotton had been continuously cultivated for 0, 1, 3, and 5 years. Rhizosphere soil
Hai-Yan Hu; Xiao-Xia Liu; Zhang-Wu Zhao; Jian-Guang Sun; Qing-Wen Zhang; Xing-Zhong Liu; Yong Yu
Silibinin and Paclitaxel Cotreatment Significantly Suppress the Activity and Lung Metastasis of Triple Negative 4T1 Mammary Tumor Cell in Mice
The in vitro and in vivo bioactivities of silibinin (SB), paclitaxel (PTX) and SB and PTX in combination (SB+PTX) against murine metastatic mammary 4T1 cancer cell line were investigated. Isobologram and combination index (CI) analyses showed that SB and PTX can function synergistically in the inhibition of 4T1 cell proliferation with a CI value < 1. Both SB and PTX alone or SB+PTX treatment inhibited 4T1 cell migration and motility possibly through downregulation of the serpin protease nexin-1 (PN-1) and N-cadherin expression, inhibition of matrix metalloprotease (MMP)-9 activity, and upregulation of E-cadherin. Flow cytometry and Western blot analyses demonstrated that both drugs deregulated cell-cycle mediators and induced apoptosis in 4T1 cells. A real-time in vivo bioluminescence imaging system to monitor the breast cancer cell metastasis in syngeneic BALB/c mice was established using a stable 4T1pGL-COX-2/Luc cell clone carrying a COX-2 promoter driven-luciferase reporter gene. In vivo study using the allograft 4T1pGL-COX-2/Luc metastatic mouse model indicated that SB co-treated with PTX can significantly suppress lung metastasis of 4T1 cells likely through inhibiting cell proliferation and angiogenesis. Together, this study demonstrates that SB could act synergistically with PTX in 4T1 cells, providing a therapeutic option for highly metastatic triple negative breast cancer.
Ho, Bing-Ying; Lin, Chun-Hung; Apaya, Maria Karmella; Chao, Wen-Wan; Shyur, Lie-Fen
An increase in melatonin in transgenic rice causes pleiotropic phenotypes, including enhanced seedling growth, delayed flowering, and low grain yield.
No previous reports have described the effects of an increase in endogenous melatonin levels on plant yield and reproduction. Here, the phenotypes of melatonin-rich transgenic rice plants overexpressing sheep serotonin N-acetyltransferase were investigated under field conditions. Early seedling growth of melatonin-rich transgenic rice was greatly accelerated, with enhanced biomass relative to the wild type (WT). However, flowering was delayed by 1 wk in the transgenic lines compared with the WT. Grain yields of the melatonin-rich transgenic lines were reduced by 33% on average. Other phenotypes also varied among the transgenic lines. For example, the transgenic line S1 exhibited greater height and biomass than the WT, while the S10 transgenic line showed diminished height and an increase in panicle numbers per plant. The expression levels of Oryza sativa homeobox1 (OSH1) and TEOSINTE BRANCHED1 (TB1) genes, two key regulators of meristem initiation and maintenance, were not altered in the transgenic lines. These data demonstrate that an alteration of endogenous melatonin levels leads to pleiotropic effects such as height, biomass, panicle number, flowering time, and grain yield, indicating that melatonin behaves as a signaling molecule in plant growth and reproduction. PMID:24571270
Byeon, Yeong; Back, Kyoungwhan
Transgenic Mosquitoes Expressing a Phospholipase A2 Gene Have a Fitness Advantage When Fed Plasmodium falciparum-Infected Blood
Background Genetically modified mosquitoes have been proposed as an alternative strategy to reduce the heavy burden of malaria. In recent years, several proof-of-principle experiments have been performed that validate the idea that mosquitoes can be genetically modified to become refractory to malaria parasite development. Results We have created two transgenic lines of Anophelesstephensi, a natural vector of Plasmodium falciparum, which constitutively secrete a catalytically inactive phospholipase A2 (mPLA2) into the midgut lumen to interfere with Plasmodium ookinete invasion. Our experiments show that both transgenic lines expressing mPLA2 significantly impair the development of rodent malaria parasites, but only one line impairs the development of human malaria parasites. In addition, when fed on malaria-infected blood, mosquitoes from both transgenic lines are more fecund than non-transgenic mosquitoes. Consistent with these observations, cage experiments with mixed populations of transgenic and non-transgenic mosquitoes show that the percentage of transgenic mosquitoes increases when maintained on Plasmodium-infected blood. Conclusions Our results suggest that the expression of an anti-Plasmodium effector gene gives transgenic mosquitoes a fitness advantage when fed malaria-infected blood. These findings have important implications for future applications of transgenic mosquito technology in malaria control.
Smith, Ryan C.; Kizito, Christopher; Rasgon, Jason L.; Jacobs-Lorena, Marcelo
Development of transgenic animals for optogenetic manipulation of mammalian nervous system function: progress and prospects for behavioral neuroscience.
Here we review the rapidly growing toolbox of transgenic mice and rats that exhibit functional expression of engineered opsins for neuronal activation and silencing with light. Collectively, these transgenic animals are enabling neuroscientists to access and manipulate the many diverse cell types in the mammalian nervous system in order to probe synaptic and circuitry connectivity, function, and dysfunction. The availability of transgenic lines affords important advantages such as stable and heritable transgene expression patterns across experimental cohorts. As such, the use of transgenic lines precludes the need for other costly and labor-intensive procedures to achieve functional transgene expression in each individual experimental animal. This represents an important consideration when large cohorts of experimental animals are desirable as in many common behavioral assays. We describe the diverse strategies that have been implemented for developing transgenic mouse and rat lines and highlight recent advances that have led to dramatic improvements in achieving functional transgene expression of engineered opsins. Furthermore, we discuss considerations and caveats associated with implementing recently developed transgenic lines for optogenetics-based experimentation. Lastly, we propose strategies that can be implemented to develop and refine the next generation of genetically modified animals for behaviorally-focused optogenetics-based applications. PMID:23473879
Ting, Jonathan T; Feng, Guoping
MYMIV-AC2, a geminiviral RNAi suppressor protein, has potential to increase the transgene expression.
Gene silencing is one of the limiting factors for transgene expression in plants. But the plant viruses have learnt to suppress gene silencing by encoding the protein(s), called RNA silencing suppressor(s) (RSS). Hence, these proteins could be used to overcome the limitation for transgene expression. The RNAi suppressors, namely HC-Pro and P19, have been shown to enhance the transgene expression but other RSS proteins have not been screened for similar role. Moreover, none of RSSs from the DNA viruses are known for enhancing the expression of transgenes. The Mungbean Yellow Mosaic India Virus (MYMIV) belonging to the genus Begomovirus within the family of Geminiviridae encodes an RSS called the AC2 protein. Here, we used AC2 to elevate the expression of the transgenes. Upon introduction of MYMIV-AC2 in the silenced GFP transgenic tobacco lines, by either genetic hybridisation or transgenesis, the GFP expression was enhanced several fold in F1 and T0 lines. The GFP-siRNA levels were much reduced in F1 and T0 lines compared with those of the initial parental silenced lines. The enhanced GFP expression was also observed at the cellular level. This approach was also successful in enhancing the expression of another transgene, namely topoisomeraseII. PMID:22592775
Rahman, Jamilur; Karjee, Sumona; Mukherjee, Sunil Kumar
Detection of Sweet and Umami Taste in the Absence of Taste Receptor T1r3
The tastes of sugars (sweet) and glutamate (umami) are thought to be detected by T1r receptors expressed in taste cells. Molecular genetics and heterologous expression implicate T1r2 plus T1r3 as a sweet-responsive receptor, and T1r1 plus T1r3, as well as a truncated form of the type 4 metabotropic glutamate receptor (taste-mGluR4), as umami-responsive receptors. Here, we show that mice lacking
Sami Damak; Minqing Rong; Keiko Yasumatsu; Zaza Kokrashvili; Vijaya Varadarajan; Shiying Zou; Peihua Jiang; Yuzo Ninomiya; Robert F. Margolskee
Transgene x Environment Interactions in Genetically Modified Wheat
Background The introduction of transgenes into plants may cause unintended phenotypic effects which could have an impact on the plant itself and the environment. Little is published in the scientific literature about the interrelation of environmental factors and possible unintended effects in genetically modified (GM) plants. Methods and Findings We studied transgenic bread wheat Triticum aestivum lines expressing the wheat Pm3b gene against the fungus powdery mildew Blumeria graminis f.sp. tritici. Four independent offspring pairs, each consisting of a GM line and its corresponding non-GM control line, were grown under different soil nutrient conditions and with and without fungicide treatment in the glasshouse. Furthermore, we performed a field experiment with a similar design to validate our glasshouse results. The transgene increased the resistance to powdery mildew in all environments. However, GM plants reacted sensitive to fungicide spraying in the glasshouse. Without fungicide treatment, in the glasshouse GM lines had increased vegetative biomass and seed number and a twofold yield compared with control lines. In the field these results were reversed. Fertilization generally increased GM/control differences in the glasshouse but not in the field. Two of four GM lines showed up to 56% yield reduction and a 40-fold increase of infection with ergot disease Claviceps purpurea compared with their control lines in the field experiment; one GM line was very similar to its control. Conclusions Our results demonstrate that, depending on the insertion event, a particular transgene can have large effects on the entire phenotype of a plant and that these effects can sometimes be reversed when plants are moved from the glasshouse to the field. However, it remains unclear which mechanisms underlie these effects and how they may affect concepts in molecular plant breeding and plant evolutionary ecology.
Zeller, Simon L.; Kalinina, Olena; Brunner, Susanne; Keller, Beat; Schmid, Bernhard
CgT1: a non-LTR retrotransposon with restricted distribution in the fungal phytopathogen Colletotrichum gloeosporioides.
Two genetically distinct biotypes (A and B) of Colletotrichum gloeosporioides that cause different anthracnose diseases on the legumes Stylosanthes spp. have been identified in Australia. A DNA sequence that was present in biotype B and absent in biotype A was isolated by differential hybridisation of a genomic library using total genomic DNA of each biotype as hybridisation probes. This sequence also failed to hybridise to DNA of three biotypes of C. gloeosporioides from other host species and to DNA of three other species of Colletotrichum. This clone was used to isolate two cosmid clones of biotype B. Sequence analysis of these clones revealed a repetitive element of approximately 5.7 kb in length. This element, termed CgT1, was dispersed in the genome and present in about 30 copies. The element contained open reading frames encoding deduced sequence motifs homologous to gag-like proteins, reverse transcriptase and RNase H domains of non-LTR retrotransposons. The termini of CgT1 lacked long terminal repeats (LTRs) but contained a 3' A-rich domain. The insertion site of one copy of the element was flanked by short 13-bp direct repeats. These characteristics of the termini, taken together with the overall structure and sequence homologies, indicate that CgT1 belongs to the non-LTR, LINE-like retrotransposon class of elements that are present in many eukaryotes. PCR primers designed to amplify regions of CgT1 can be used to distinguish biotypes A and B in Australia. DNA fingerprinting analysis of genomic DNA using hybridisation probes derived from the terminal regions of CgT1 revealed that Australian isolates of biotype B are monomorphic. CgT1 was not detected in some isolates causing Type B disease from other countries and when CgT1 was present there was considerable polymorphism in CgT1 organisation in the genome. CgT1 is the first transposon-like element to be identified in the genus Colletotrichum and has considerable potential as a tool for the study of population structure, genome dynamics and evolution in C. gloeosporioides. PMID:8842152
He, C; Nourse, J P; Kelemu, S; Irwin, J A; Manners, J M
A transgenic mouse that expresses a diversity of human sequence heavy and light chain immunoglobulins.
We have generated transgenic mice that express a diverse repertoire of human sequence immunoglobulins. The expression of this repertoire is directed by light and heavy chain minilocus transgenes comprised of human protein coding sequences in an unrearranged, germ-line configuration. In this paper we describe the construction of these miniloci and the composition of the CDR3 repertoire generated by the transgenic mice. The largest transgene discussed is a heavy chain minilocus that includes human mu and gamma 1 coding sequences together with their respective switch regions. It consists of a single 61 kb DNA fragment propagated in a bacterial plasmid vector. Both human heavy chain classes are expressed in animals that carry the transgene. In light chain transgenic animals the unrearranged minilocus sequences recombine to form VJ joints that use all five human J kappa segments, resulting in a diversity of human-like CDR3 regions. Similarly, in heavy chain transgenics the inserted sequences undergo VDJ joining complete with N region addition to generate a human-like VH CDR3 repertoire. All six human JH segments and at least eight of the ten transgene encoded human D segments are expressed. The transgenic animals described in this paper represent a potential source of human sequence antibodies for in vivo therapeutic applications.
Taylor, L D; Carmack, C E; Schramm, S R; Mashayekh, R; Higgins, K M; Kuo, C C; Woodhouse, C; Kay, R M; Lonberg, N
[Traumatic fracture of the thoracic spine T1-T10].
We describe the incidence, causes, management and prognosis of traumatic fractures of the thoracic spine from T1 to T10 in surgical cases of traumatic fractures of spine during the period from June 1994 to June 2003 studied retrospectively. The type of fracture was determined according to the Gertzbein classification, and the degree of stability using the Denis classification. The neurological picture at admission and 30 days after surgery was evaluated using the ASIA/IMSOP classification. Surgery was performed in patients with complete spinal cord injury (n=7) for the purpose of stabilization using the posterior approach. In cases without spinal cord injury or incomplete injury (n=12), the surgical procedure was performed aiming to decompress the nerve tissue, to correct the alignment of the spine and to stabilize the spine. PMID:15608977
Falavigna, Asdrubal; Righesso Neto, Orlando; Ferraz, Fernando Antonio Patriani; Boniatti, Mßrcio Manozzo
Effect of Air Ions on Submicron T1 Bacteriophage Aerosols
The effect of a high concentration of ionized air molecules on sampling T1 phage aerosols of submicron particle size was evaluated by comparing the phage recoveries of all-glass impingers (AGI-4) and type 6 filter papers. Sampler recoveries of all ionized aerosols were less than the recoveries of nonionized control aerosols. These reductions in recovery were greater with positive ions than with negative ions or ions of mixed polarity. The AGI-4 allowed considerable slippage, which was not affected by the air ions. Type 6 filter paper recoveries were less than AGI-4 recoveries. The air ions did not appear to affect the aerosol particle size as determined by an electron microscope. Images Fig. 1 Fig. 3
Happ, John W.; Harstad, J. Bruce; Buchanan, Lee M.
Pairing symmetry signatures of T1 in superconducting ferromagnets
NASA Astrophysics Data System (ADS)
We study the nuclear relaxation rate 1/T1 as a function of temperature for a superconducting ferromagnetic coexistent system using a p -wave triplet model for the superconducting pairing symmetry. This calculation is contrasted with a singlet s -wave previously studied. We see for the s -wave case that there is a Hebel-Slichter peak, albeit reduced due to the magnetization, and no peak for the p -wave case. We then compare these results to a nuclear relaxation rate experiment on UGe2 to determine the possible pairing symmetry signatures in that material. It is seen that the experimental data are inconclusive to rule out the possibility of s -wave pairing in UGe2 .
Dahal, Hari P.; Jackiewicz, Jason; Bedell, Kevin S.
The ability of Papaya ringspot virus strains overcoming the transgenic resistance of papaya conferred by the coat protein gene is not correlated with higher degrees of sequence divergence from the transgene
The coat protein (CP) gene mediated transgenic resistance is found to be the best approach for protecting papaya plants against the destructive disease caused by Papaya ringspot viruses(PRSV). In order to study the variability of PRSV and the potential threat to the CP-transgenic resistance, five virus isolates were collected from transgenic plants of papaya line 16-0-1, which carry the CP
Savarni Tripathi; Huey-jiunn Bau; Li-fang Chen; Shyi-dong Yeh
Synthetic versions of firefly luciferase and Renilla luciferase reporter genes that resist transgene silencing in sugarcane
Background Down-regulation or silencing of transgene expression can be a major hurdle to both molecular studies and biotechnology applications in many plant species. Sugarcane is particularly effective at silencing introduced transgenes, including reporter genes such as the firefly luciferase gene. Synthesizing transgene coding sequences optimized for usage in the host plant is one method of enhancing transgene expression and stability. Using specified design rules we have synthesised new coding sequences for both the firefly luciferase and Renilla luciferase reporter genes. We have tested these optimized versions for enhanced levels of luciferase activity and for increased steady state luciferase mRNA levels in sugarcane. Results The synthetic firefly luciferase (luc*) and Renilla luciferase (Renluc*) coding sequences have elevated G?+?C contents in line with sugarcane codon usage, but maintain 75% identity to the native firefly or Renilla luciferase nucleotide sequences and 100% identity to the protein coding sequences. Under the control of the maize pUbi promoter, the synthetic luc* and Renluc* genes yielded 60x and 15x higher luciferase activity respectively, over the native firefly and Renilla luciferase genes in transient assays on sugarcane suspension cell cultures. Using a novel transient assay in sugarcane suspension cells combining co-bombardment and qRT-PCR, we showed that synthetic luc* and Renluc* genes generate increased transcript levels compared to the native firefly and Renilla luciferase genes. In stable transgenic lines, the luc* transgene generated significantly higher levels of expression than the native firefly luciferase transgene. The fold difference in expression was highest in the youngest tissues. Conclusions We developed synthetic versions of both the firefly and Renilla luciferase reporter genes that resist transgene silencing in sugarcane. These transgenes will be particularly useful for evaluating the expression patterns conferred by existing and newly isolated promoters in sugarcane tissues. The strategies used to design the synthetic luciferase transgenes could be applied to other transgenes that are aggressively silenced in sugarcane.
Transgenic plants expressing HC-Pro show enhanced virus sensitivity while silencing of the transgene results in resistance.
Nicotiana benthamiana plants were engineered to express sequences of the helper component-proteinase (HC-Pro) of Cowpea aphid-borne mosaic potyvirus (CABMV). The sensitivity of the transgenic plants to infection with parental and heterologous viruses was studied. The lines expressing HC-Pro showed enhanced symptoms after infection with the parental CABMV isolate and also after infection with a heterologous potyvirus, Potato virus Y (PVY) and a comovirus, Cowpea mosaic virus (CPMV). On the other hand, transgenic lines expressing nontranslatable HC-Pro or translatable HC-Pro with a deletion of the central domain showed wild type symptoms after infection with the parental CABMV isolate and heterologous viruses. These results showed that CABMV HC-Pro is a pathogenicity determinant that conditions enhanced sensitivity to virus infection in plants, and that the central domain of the protein is essential for this. The severe symptoms in CABMV-infected HC-Pro expressing lines were remarkably followed by brief recovery and subsequent re-establishment of infection, possibly indicating counteracting effects of HC-Pro expression and a host defense response. One of the HC-Pro expressing lines (h48) was found to contain low levels of transgenic HC-Pro RNA and to be resistant to CABMV and to recombinant CPMV expressing HC-Pro. This indicated that h48 was (partially) posttranscriptionally silenced for the HC-Pro transgene inspite of the established role of HC-Pro as a suppressor of posttranscriptional gene silencing. Line h48 was not resistant to PVY, but instead showed enhanced symptoms compared to nontransgenic plants. This may be due to relief of silencing of the HC-Pro transgene by HC-Pro expressed by PVY. PMID:12206307
Mlotshwa, Sizolwenkosi; Verver, Jan; Sithole-Niang, Idah; Prins, Marcel; Van Kammen, A B; Wellink, Joan
14N quadrupole resonance and 1H T1 dispersion in the explosive RDX.
The explosive hexahydro-1,3,5-trinitro-s-triazine (CH2-N-NO2)3, commonly known as RDX, has been studied by 14N NQR and 1H NMR. NQR frequencies and relaxation times for the three ?+ and ?- lines of the ring 14N nuclei have been measured over the temperature range 230-330 K. The 1H NMR T1 dispersion has been measured for magnetic fields corresponding to the 1H NMR frequency range of 0-5.4 M Hz. The results have been interpreted as due to hindered rotation of the NO2 group about the N-NO2 bond with an activation energy close to 92 kJ mol(-1). Three dips in the 1H NMR dispersion near 120, 390 and 510 kHz are assigned to the ?0, ?- and ?+ transitions of the 14NO2 group. The temperature dependence of the inverse line-width parameters T2? of the three ?+ and ?- ring nitrogen transitions between 230 and 320 K can be explained by a distribution in the torsional oscillational amplitudes of the NO2 group about the N-NO2 bond at crystal defects whose values are consistent with the latter being mainly edge dislocations or impurities in the samples studied. Above 310 K, the 14N line widths are dominated by the rapid decrease in the spin-spin relaxation time T2 due to hindered rotation of the NO2 group. A consequence of this is that above this temperature, the 1H T1 values at the quadrupole dips are dominated by the spin mixing time between the 1H Zeeman levels and the combined 1H and 14N spin-spin levels. PMID:21978662
Smith, John A S; Blanz, Martin; Rayner, Timothy J; Rowe, Michael D; Bedford, Simon; Althoefer, Kaspar
Diurnal changes of arginine vasopressin-enhanced green fluorescent protein fusion transgene expression in the rat suprachiasmatic nucleus
We have recently developed a new transgenic rat line expressing an arginine vasopressin (AVP)-enhanced green fluorescent protein (eGFP) fusion gene. The AVP-eGFP transgene is expressed in the paraventricular (PVN) and supraoptic (SON) nuclei and the suprachiasmatic nucleus (SCN) of the hypothalamus. Transgene expression in the PVN and SON showed an exaggerated response to salt loading and nociceptive stimulation. However, the
Takashi Maruyama; Toyoaki Ohbuchi; Hiroaki Fujihara; Minori Shibata; Koji Mori; David Murphy; Govindan Dayanithi; Yoichi Ueta
Testis-Specific Expression of a Metallothionein I-Driven Transgene Correlates with Undermethylation of the Locus in Testicular DNA
Mice carrying a chimeric transgene of the human testis-specific lactate dehydrogenase cDNA driven by mouse metallothionein I promoter have been reported to express the transgene in a testis-specific manner in six founder lines. To study the mechanism by which this testis-specific expression is mediated, we have examined genomic placement, expression pattern, and methylation status of the transgene. Our results indicate
Kourosh Salehi-Ashtiani; Robert J. Widrow; Clement L. Markert; Erwin Goldberg
Expression of CD4 in Transgenic Mice Alters the Specificity of CD8 Cells for Allogeneic Major Histocompatibility Complex
We have generated a transgenic mouse line in which a CD4 transgene is expressed on a significant fraction of the mature CD8^+ lymphocytes but is not expressed in the thymus. This provides an opportunity to examine the functional consequences of CD4 expression in a population of class I-selected CD8^+ lymphocytes. CD8^+ lymphocytes expressing the CD4 transgene proliferate in response to
Ellen Robey; Fred Ramsdell; James Elliott; David Raulet; Dimitris Kloussis; Richard Axel; B. J. Fowlkes
Transmission and expression of an integrated reporter construct in three generations of transgenic mud loach ( Misgurnus mizolepis)
We produced transgenic mud loach lines by microinjecting a CAT reporter gene driven by the carp ?-actin promoter into fertilized eggs and demonstrated successful integration, transmission to F3, with variable patterns of expression in different tissues. Microinjection yielded transgenic F0 individuals with a frequency that ranged from 9.3 to 27.8% among microinjected groups. Of 63 transgenic founders identified, seven males
Yoon Kwon Nam; Choong Hwan Noh; Dong Soo Kim
Eosinophil-Deficient Transgenic Animals.
National Technical Information Service (NTIS)
The technologies described herein are based on the discovery that expression of a toxin gene under control of an eosinophil-specific promoter can cause the ablation of eosinophils in a transgenic animal. Accordingly, the nucleic acid constructs featured i...
J. J. Lee, N. A. Lee
Transgenic mice in developmental toxicology.
National Technical Information Service (NTIS)
Advances in molecular biology and embryology are being utilized for the generation of transgenic mice, animals that contain specific additions, deletions, or modifications of genes or sequences in their DNA. Mouse embryonic stem cells and homologous recom...
R. P. Woychik
[Development of a hepatitis B virus carrier transgenic mice model].
The studies for the development of transgenic mice models which provide important profits for the studies concerning immunopathogenesis of hepatitis B virus (HBV) infections are in progress since 20 years. For this purpose different lineages bearing whole HBV genome or selected viral genes have been developed and their usage in clarifying the HBV replication and pathogenesis mechanisms have been emphasized. The aim of this study was to develop and breed a HBV carrier mice model. In the study the full HBV genome has been transferred to mouse embryos by microinjection procedure. Following transgenic manipulation, the HBV carriers among the daughter mice have been detected by molecular methods in which HBV-DNA replication and expression have been shown. The manipulations for transgene transfers have been performed in TUBITAK Marmara Research Center Transgene Laboratory, Gebze, Istanbul. The HBV-DNA carrier mice have been demonstrated by polymerase chain reaction (PCR) using the DNA samples obtained from tail tissues and also by dot-blot hybridization of the mice sera. Integrated HBV-DNA has been detected by applying in-situ hybridization to the liver tissue sections. HBV-DNA expression has been shown by reverse transcriptase PCR method with total RNA molecules that have been isolated from the liver tissues of the HBV-DNA carrier mice. HBsAg has been detected in the liver by immunohistochemical method, and HBsAg and HBeAg have additionally been demonstrated by ELISA. HBV genome, expression of the genome and the expression products have been determined in approximately 10% of the mice of which HBV-DNA have been transferred. By inbreeding heterozygote carrier mice, homozygote HBV transgenic mice line have been obtained. These HBV transgenic mice are the first lineages developed in our country. It is hopefully thought that this HBV carrier transgenic mouse model may contribute to the studies on the pathogenesis of HBV infections which are important health problems in the world as well as in Turkey. PMID:18444564
Caner, M³ge; Arat, Sezen; Bircan, Rifat
PsT-1: a new tryptophyllin peptide from the skin secretion of Waxy Monkey Leaf Frog, Phyllomedusa sauvagei.
The Waxy Monkey Leaf Frog, Phyllomedusa sauvagei, has been extensively-studied for many years, and a broad spectrum of bioactive peptides has been found in its skin secretions. Here we report the discovery of a novel tryptophyllin (TPH) peptide, named PsT-1, from this frog species. Skin secretions from specimens of P. sauvagei were collected by mild electrical stimulation. Peptides were identified and characterized by transcriptome cloning, and the structure was confirmed by MALDI-TOF mass spectrometry and automated Edman degradation. This novel peptide was encoded by a single precursor of 61 amino acid residues, whose primary structure was deduced from cloned skin cDNA. Analysis of different amphibian tryptophyllins revealed that PsT-1 exhibited a high degree of primary structural similarity to its homologs, PdT-1 and PdT-2, from the Mexican giant leaf frog, Pachymedusa dacnicolor. A synthetic replicate of PsT-1 was found to inhibit bradykinin-induced vasorelaxation of phenylephrine pre-constricted rat tail artery smooth muscle. It was also found that PsT-1 had an anti-proliferative effect on three different human prostate cancer cell lines (LNCaP/PC3/DU145), by use of an MTT assay coupled with direct cell counting as measures of cell growth. These data indicate that PsT-1 is a likely bradykinin receptor antagonist and its biological effects are probably mediated through bradykinin receptors. As a BK antagonist, PST-1, with antagonistic effects on BK in artery smooth muscle, inhibition of proliferation in prostate cancer cells and lack of undesirable side effects, may have potential in cardiovascular, inflammatory and anticancer therapy. PMID:23518460
Wang, Ran; Chen, Tianbao; Zhou, Mei; Wang, Lei; Shaw, Chris
Measurement of T1/T2 relaxation times in overlapped regions from homodecoupled 1H singlet signals
NASA Astrophysics Data System (ADS)
The implementation of the HOmodecoupled Band-Selective (HOBS) technique in the conventional Inversion-Recovery and CPMG-based PROJECT experiments is described. The achievement of fully homodecoupled signals allows the distinction of overlapped 1H resonances with small chemical shift differences. It is shown that the corresponding T1 and T2 relaxation times can be individually measured from the resulting singlet lines using conventional exponential curve-fitting methods.
Casta±ar, Laura; Nolis, Pau; Virgili, Albert; Parella, Teodor
Comparison between volatile emissions from transgenic apples and from two representative classically bred apple cultivars.
While most risk assessments contrast a transgenic resistant to its isogenic line, an additional comparison between the transgenic line and a classically bred cultivar with the same resistance gene would be highly desirable. Our approach was to compare headspace volatiles of transgenic scab resistant apple plants with two representative cultivars (the isogenic 'Gala' and the scab resistance gene-containing 'Florina'). As modifications in volatile profiles have been shown to alter plant relationships with non-target insects, we analysed headspace volatiles from apple plants subjected to different infection types by gas chromatography-mass spectrometry. Marked differences were found between healthy and leafminer (Phyllonorycter blancardella) infested genotypes, where emissions between the transgenic scab resistant line and the two cultivars differed quantitatively in four terpenes and an aromatic compound. However, these modified odour emissions were in the range of variability of the emissions recorded for the two standard cultivars that proved to be crucial references. PMID:19543801
Vogler, Ute; Rott, Anja S; Gessler, Cesare; Dorn, Silvia
Role of nutrient-sensing taste 1 receptor (T1R) family members in gastrointestinal chemosensing.
Luminal nutrient sensing by G-protein-coupled receptors (GPCR) expressed on the apical domain of enteroendocrine cells activates intracellular pathways leading to secretion of gut hormones that control vital physiological processes such as digestion, absorption, food intake and glucose homeostasis. The taste 1 receptor (T1R) family of GPCR consists of three members: T1R1; T1R2; T1R3. Expression of T1R1, T1R2 and T1R3 at mRNA and protein levels has been demonstrated in the intestinal tissue of various species. It has been shown that T1R2-T1R3, in association with G-protein gustducin, is expressed in intestinal K and L endocrine cells, where it acts as the intestinal glucose (sweet) sensor. A number of studies have demonstrated that activation of T1R2-T1R3 by natural sugars and artificial sweeteners leads to secretion of glucagon-like peptides 1&2 (GLP-1 and GLP-2) and glucose dependent insulinotropic peptide (GIP). GLP-1 and GIP enhance insulin secretion; GLP-2 increases intestinal growth and glucose absorption. T1R1-T1R3 combination co-expressed on the apical domain of cholecystokinin (CCK) expressing cells is a luminal sensor for a number of L-amino acids; with amino acid-activation of the receptor eliciting CCK secretion. This article focuses on the role of the gut-expressed T1R1, T1R2 and T1R3 in intestinal sweet and L-amino acid sensing. The impact of exploiting T1R2-T1R3 as a nutritional target for enhancing intestinal glucose absorption and gut structural maturity in young animals is also highlighted. PMID:24382171
Shirazi-Beechey, Soraya P; Daly, Kristian; Al-Rammahi, Miran; Moran, Andrew W; Bravo, David
Permanent Neonatal Diabetes in INSC94Y Transgenic Pigs
Mutations in the insulin (INS) gene may cause permanent neonatal diabetes mellitus (PNDM). Ins2 mutant mouse models provided important insights into the disease mechanisms of PNDM but have limitations for translational research. To establish a large animal model of PNDM, we generated INSC94Y transgenic pigs. A line expressing high levels of INSC94Y mRNA (70¢86% of wild-type INS transcripts) exhibited elevated blood glucose soon after birth but unaltered ?-cell mass at the age of 8 days. At 4.5 months, INSC94Y transgenic pigs exhibited 41% reduced body weight, 72% decreased ?-cell mass (?53% relative to body weight), and 60% lower fasting insulin levels compared with littermate controls. ?-cells of INSC94Y transgenic pigs showed a marked reduction of insulin secretory granules and severe dilation of the endoplasmic reticulum. Cataract development was already visible in 8-day-old INSC94Y transgenic pigs and became more severe with increasing age. Diabetes-associated pathological alterations of kidney and nervous tissue were not detected during the observation period of 1 year. The stable diabetic phenotype and its rescue by insulin treatment make the INSC94Y transgenic pig an attractive model for insulin supplementation and islet transplantation trials, and for studying developmental consequences of maternal diabetes mellitus.
Renner, Simone; Braun-Reichhart, Christina; Blutke, Andreas; Herbach, Nadja; Emrich, Daniela; Streckel, Elisabeth; Wunsch, Annegret; Kessler, Barbara; Kurome, Mayuko; Bahr, Andrea; Klymiuk, Nikolai; Krebs, Stefan; Puk, Oliver; Nagashima, Hiroshi; Graw, Jochen; Blum, Helmut; Wanke, Ruediger; Wolf, Eckhard
Bioaccessibility of carotenoids from transgenic provitamin A biofortified sorghum.
Biofortified sorghum (Sorghum bicolor (L.) Moench) lines are being developed to target vitamin A deficiency in Sub-Saharan Africa, but the delivery of provitamin A carotenoids from such diverse germplasms has not been evaluated. The purpose of this study was to screen vectors and independent transgenic events for the bioaccessibility of provitamin A carotenoids using an in vitro digestion model. The germplasm background and transgenic sorghum contained 1.0-1.5 and 3.3-14.0 ?g/g ?-carotene equivalents on a dry weight basis (DW), respectively. Test porridges made from milled transgenic sorghum contained up to 250 ?g of ?-carotene equivalents per 100 g of porridge on a fresh weight basis (FW). Micellarization efficiency of all-trans-?-carotene was lower (p < 0.05) from transgenic sorghum (1-5%) than from null/nontransgenic sorghum (6-11%) but not different between vector constructs. Carotenoid bioaccessibility was significantly improved (p < 0.05) by increasing the amount of coformulated lipid in test porridges from 5% w/w to 10% w/w. Transgenic sorghum event Homo188-A contained the greatest bioaccessible ?-carotene content, with a 4-8-fold increase from null/nontransgenic sorghum. While the bioavailability and bioconversion of provitamin A carotenoids from these grains must be confirmed in vivo, these data support the notion that biofortification of sorghum can enhance total and bioaccessible provitamin A carotenoid levels. PMID:23692305
Lipkie, Tristan E; De Moura, Fabiana F; Zhao, Zuo-Yu; Albertsen, Marc C; Che, Ping; Glassman, Kimberly; Ferruzzi, Mario G
Pharming and transgenic plants.
Plant represented the essence of pharmacopoeia until the beginning of the 19th century when plant-derived pharmaceuticals were partly supplanted by drugs produced by the industrial methods of chemical synthesis. In the last decades, genetic engineering has offered an alternative to chemical synthesis, using bacteria, yeasts and animal cells as factories for the production of therapeutic proteins. More recently, molecular farming has rapidly pushed towards plants among the major players in recombinant protein production systems. Indeed, therapeutic protein production is safe and extremely cost-effective in plants. Unlike microbial fermentation, plants are capable of carrying out post-translational modifications and, unlike production systems based on mammalian cell cultures, plants are devoid of human infective viruses and prions. Furthermore, a large panel of strategies and new plant expression systems are currently developed to improve the plant-made pharmaceutical's yields and quality. Recent advances in the control of post-translational maturations in transgenic plants will allow them, in the near future, to perform human-like maturations on recombinant proteins and, hence, make plant expression systems suitable alternatives to animal cell factories. PMID:17875476
Liķnard, David; Sourrouille, Christophe; Gomord, Vķronique; Faye, Lo'c
Station-generated impulse noise and its effects on a T1 carrier signal
NASA Astrophysics Data System (ADS)
Studies dating back to 1963 emphasize the importance of considering the effects of impulse noise coupled by near end crosstalk (NEXT) as a predominant source of digital transmission error in T1 systems. Relays and switches, rectifier power supplies, ac power wiring, lines carrying test tons, and ringing generators in central offices and remote terminals have all been implicated as impulse noise sources. This study demonstrates that station-generated impulse noise can also be a problem. For instance, when the ringing signal is interrupted on a voice pair in the same cable, the disturbances can be at least 20 dB stronger than intersystem NEXT. While such strong impulses are infrequent, they may be of concern as the DS1 service to customer premises continues to expand. A simple, parallel R-L filter placed in series with a standard telephone set effectively reduce errors in the transmitted data.
Haber, J. B.
Transgene- and locus-dependent imprinting reveals allele-specific chromosome conformations
When positioned into the integrin ?-6 gene, an Hoxd9lacZ reporter transgene displayed parental imprinting in mouse embryos. While the expression from the paternal allele was comparable with patterns seen for the same transgene when present at the neighboring HoxD locus, almost no signal was scored at this integration site when the transgene was inherited from the mother, although the Itga6 locus itself is not imprinted. The transgene exhibited maternal allele-specific DNA hypermethylation acquired during oogenesis, and its expression silencing was reversible on passage through the male germ line. Histone modifications also corresponded to profiles described at known imprinted loci. Chromosome conformation analyses revealed distinct chromatin microarchitectures, with a more compact structure characterizing the maternally inherited repressed allele. Such genetic analyses of well-characterized transgene insertions associated with a de novo-induced parental imprint may help us understand the molecular determinants of imprinting.
Lonfat, Nicolas; Montavon, Thomas; Jebb, David; Tschopp, Patrick; Nguyen Huynh, Thi Hanh; Zakany, Jozsef; Duboule, Denis
Pronuclear injection-based mouse targeted transgenesis for reproducible and highly efficient transgene expression
Mouse transgenesis has proven invaluable for analysis of gene function and generation of human disease models. We describe here the development of a pronuclear injection-based targeted transgenesis (PITT) system, involving site-specific integration in fertilized eggs. The system was applied to two different genomic target loci to generate a series of transgenic lines including fluorescent mice, which reproducibly displayed strong, ubiquitous and stable transgene expression. We also demonstrated that knockdown mice could be readily generated by PITT by taking advantage of the reproducible and highly efficient expression system. The PITT system, which circumvents the problem of unpredictable and unstable transgene expression of conventional random-integration transgenic mice, reduces the time, cost and effort needed to generate transgenic mice, and is potentially applicable to both in vivo ægain-of-functionÆ and æloss-of-functionÆ studies.
Ohtsuka, Masato; Ogiwara, Sanae; Miura, Hiromi; Mizutani, Akiko; Warita, Takayuki; Sato, Masahiro; Imai, Kenji; Hozumi, Katsuto; Sato, Takehito; Tanaka, Masafumi; Kimura, Minoru; Inoko, Hidetoshi
Development of Hyperplasias, Preneoplasias, and Mammary Tumors in MMTV-c-erbB-2 and MMTV-TGF? Transgenic Rats
Human cDNAs corresponding to two epidermal growth factor-related products that are overexpressed in human breast cancers, that for c-erbB-2 (HER-2) and for transforming growth factor ? (TGF?), have been cloned downstream of the mouse mammary tumor virus (MMTV) long terminal repeat promoter and injected into the pronucleus of fertilized oocytes of Sprague-Dawley rats to produce transgenic offspring. Expression of the transgenic mRNAs is not detectable in mammary tissue from virgin transgenic rats but is detected in mammary tissue from certain lines of mid-pregnant transgenic rats. When two such lines of either type of transgenic rat are subjected to repeated cycles of pregnancy and lactation, they produce, primarily in the mammary glands, extensive pathologies, whereas virgin transgenic rats produce no such abnormalities. Multiparous transgenic female offspring from c-erbB-2-expressing lines develop a variety of focal hyperplastic and benign lesions that resemble lesions commonly found in human breasts. These lesions include lobular and ductal hyperplasia, fibroadenoma, cystic expansions, and papillary adenomas. More malignant lesions, including ductal carcinoma in situ and carcinoma, also develop stochastically at low frequency. The mammary glands of transgenic females invariably fail to involute fully after lactation. Similar phenotypes are observed in female MMTV-TGF? transgenic rats. In addition, multiparous TGF?-expressing female transgenics frequently develop severe pregnancy-dependent lactating hyperplasias as well as residual lobules of hyperplastic secretory epithelium and genuine lactating adenomas after weaning. These transgenic rat models confirm the conclusions reached in transgenic mice that overexpression of the c-erbB-2 and TGF? genes predisposes the mammary gland to stochastic tumor development.
Davies, Barry R.; Platt-Higgins, Angela M.; Schmidt, Gunter; Rudland, Philip S.
Endoscopic and surgical resection of T1a/T1b esophageal neoplasms: A systematic review
AIM: To investigate potential therapeutic recommendations for endoscopic and surgical resection of T1a/T1b esophageal neoplasms. METHODS: A thorough search of electronic databases MEDLINE, Embase, Pubmed and Cochrane Library, from 1997 up to January 2011 was performed. An analysis was carried out, pooling the effects of outcomes of 4241 patients enrolled in 80 retrospective studies. For comparisons across studies, each reporting on only one endoscopic method, we used a random effects meta-regression of the log-odds of the outcome of treatment in each study. ōNeural networksö as a data mining technique was employed in order to establish a prediction model of lymph node status in superficial submucosal esophageal carcinoma. Another data mining technique, the ōfeature selection and root cause analysisö, was used to identify the most important predictors of local recurrence and metachronous cancer development in endoscopically resected patients, and lymph node positivity in squamous carcinoma (SCC) and adenocarcinoma (ADC) separately in surgically resected patients. RESULTS: Endoscopically resected patients: Low grade dysplasia was observed in 4% of patients, high grade dysplasia in 14.6%, carcinoma in situ in 19%, mucosal cancer in 54%, and submucosal cancer in 16% of patients. There were no significant differences between endoscopic mucosal resection and endoscopic submucosal dissection (ESD) for the following parameters: complications, patients submitted to surgery, positive margins, lymph node positivity, local recurrence and metachronous cancer. With regard to piecemeal resection, ESD performed better since the number of cases was significantly less [coefficient: -7.709438, 95%CI: (-11.03803, -4.380844), P < 0.001]; hence local recurrence rates were significantly lower [coefficient: -4.033528, 95%CI: (-6.151498, -1.915559), P < 0.01]. A higher rate of esophageal stenosis was observed following ESD [coefficient: 7.322266, 95%CI: (3.810146, 10.83439), P < 0.001]. A significantly greater number of SCC patients were submitted to surgery (log-odds, ADC: -2.1206 ▒ 0.6249 vs SCC: 4.1356 ▒ 0.4038, P < 0.05). The odds for re-classification of tumor stage after endoscopic resection were 53% and 39% for ADC and SCC, respectively. Local tumor recurrence was best predicted by grade 3 differentiation and piecemeal resection, metachronous cancer development by the carcinoma in situ component, and lymph node positivity by lymphovascular invasion. With regard to surgically resected patients: Significant differences in patients with positive lymph nodes were observed between ADC and SCC [coefficient: 1.889569, 95%CI: (0.3945146, 3.384624), P < 0.01). In contrast, lymphovascular and microvascular invasion and grade 3 patients between histologic types were comparable, the respective rank order of the predictors of lymph node positivity was: Grade 3, lymphovascular invasion (L+), microvascular invasion (V+), submucosal (Sm) 3 invasion, Sm2 invasion and Sm1 invasion. Histologic type (ADC/SCC) was not included in the model. The best predictors for SCC lymph node positivity were Sm3 invasion and (V+). For ADC, the most important predictor was (L+). CONCLUSION: Local tumor recurrence is predicted by grade 3, metachronous cancer by the carcinoma in-situ component, and lymph node positivity by L+. T1b cancer should be treated with surgical resection.
Sgourakis, George; Gockel, Ines; Lang, Hauke
Sensing of amino acids by the gut-expressed taste receptor T1R1-T1R3 stimulates CCK secretion.
CCK is secreted by endocrine cells of the proximal intestine in response to dietary components, including amino acids. CCK plays a variety of roles in digestive processes, including inhibition of food intake, consistent with a role in satiety. In the lingual epithelium, the sensing of a broad spectrum of L-amino acids is accomplished by the heteromeric amino acid (umami) taste receptor (T1R1-T1R3). T1R1 and T1R3 subunits are also expressed in the intestine. A defining characteristic of umami sensing by T1R1-T1R3 is its potentiation by IMP or GMP. Furthermore, T1R1-T1R3 is not activated by Trp. We show here that, in response to L-amino acids (Phe, Leu, Glu, and Trp), but not D-amino acids, STC-1 enteroendocrine cells and mouse proximal small intestinal tissue explants secrete CCK and that IMP enhances Phe-, Leu-, and Glu-induced, but not Trp-induced, CCK secretion. Furthermore, small interfering RNA inhibition of T1R1 expression in STC-1 cells results in significant diminution of Phe-, Leu-, and Glu-stimulated, but not Trp-stimulated, CCK release. In STC-1 cells and mouse intestine, gurmarin inhibits Phe-, Leu-, and Glu-induced, but not Trp-stimulated, CCK secretion. In contrast, the Ca(2+)-sensing receptor antagonist NPS2143 inhibits Phe-stimulated CCK release partially and Trp-induced CCK secretion totally in mouse intestine. However, NPS2143 has no effect on Leu- or Glu-induced CCK secretion. Collectively, our data demonstrate that functional characteristics and cellular location of the gut-expressed T1R1-T1R3 support its role as a luminal sensor for Phe-, Leu-, and Glu-induced CCK secretion. PMID:23203156
Daly, Kristian; Al-Rammahi, Miran; Moran, Andrew; Marcello, Marco; Ninomiya, Yuzo; Shirazi-Beechey, Soraya P
Broad-Spectrum Resistance to Different Geographic Strains of Papaya ringspot virus in Coat Protein Gene Transgenic Papaya.
ABSTRACT Papaya ringspot virus (PRSV) is a major limiting factor for cultivation of papaya (Carica papaya) in tropical and subtropical areas throughout the world. Although the coat protein (CP) gene of PRSV has been transferred into papaya by particle bombardment and transgenic lines with high resistance to Hawaii strains have been obtained, they are susceptible to PRSV isolates outside of Hawaii. This strain-specific resistance limits the application of the transgenic lines in other areas of the world. In this investigation, the CP gene of a local strain isolated from Taiwan, designated PRSV YK, was transferred into papaya via Agrobacterium-mediated transformation. A total of 45 putative transgenic lines were obtained and the presence of the transgene in papaya was confirmed by polymerase chain reaction amplification. When the plants of transgenic lines were challenged with PRSV YK by mechanical inoculation, they showed different levels of resistance ranging from delay of symptom development to complete immunity. Molecular analysis of nine selected lines that exhibited different levels of resistance revealed that the expression level of the transgene is negatively correlated with the degree of resistance, suggesting that the resistance is manifested by a RNA-mediated mechanism. The segregation analysis showed that the transgene in the immune line 18-0-9 has an inheritance of two dominant loci and the other four highly resistant lines have a single dominant locus. Seven selected lines were tested further for resistance to three PRSV heterologous strains that originated in Hawaii, Thailand, and Mexico. Six of the seven lines showed varying degrees of resistance to the heterologous strains, and one line, 19-0-1, was immune not only to the homologous YK strain but also to the three heterologous strains. Thus, these CP-transgenic papaya lines with broad-spectrum resistance have great potential for use in Taiwan and other geographic areas to control PRSV. PMID:18944164
Bau, Huey-Jiunn; Cheng, Ying-Huey; Yu, Tsong-Ann; Yang, Jiu-Sherng; Yeh, Shyi-Dong
Population dynamics of Sesamia inferens on transgenic rice expressing Cry1Ac and CpTI in southern China.
Genetically modified insect-resistant rice lines containing the cry1Ac gene from Bacillus thuringiensis (Bt) or the CpTI (cowpea trypsin inhibitor) gene developed for the management of lepidopterous pests are highly resistant to the major target pests, Chilo suppressalis (Walker), Cnaphalocrocis medinalis (Guenķe), and Scirpophaga incertulas (Walker), in the main rice-growing areas of China. However, the effects of these transgenic lines on Sesamia inferens (Walker), an important lepidopterous rice pest, are currently unknown. Because different insect species have varying susceptibility to Bt insecticidal proteins that may affect population dynamics, research into the effects of these transgenic rice lines on the population dynamics of S. inferens was conducted in Fuzhou, southern China, in 2005 and 2006. The results of laboratory, field cage, and field plot experiments show that S. inferens has comparatively high susceptibility to the transgenic line during the early growing season, with significant differences observed in larval density and infestation levels between transgenic and control lines. Because of a decrease in Cry1Ac levels in the plant as it ages, the transgenic line provided only a low potential for population suppression late in the growing season. There is a correlation between the changing expression of Cry1Ac and the impact of transgenic rice on the population dynamics of S. inferens during the season. These results indicate that S. inferens may become a major pest in fields of prospective commercially released transgenic rice, and more attention should be paid to developing an effective alternative management strategy. PMID:19036217
Han, Lanzhi; Liu, Peilei; Wu, Kongming; Peng, Yufa; Wang, Feng
Transgenic mouse model of malignant skin melanoma.
Tyr-SV40E transgenic mice are specifically susceptible to melanoma due to expression of the oncogene in pigment cells. Mice of the more susceptible lines die young of early-onset eye melanomas, when skin melanomas are still infrequent and benign. To surmount this obstacle, skin from donors of two high-susceptibility lines was grafted to Tyr-SV40E hosts of a low-susceptibility line of the same inbred strain, thereby enabling the skin to outlive the donors and continue to grow in immunocompetent but tolerant hosts. Unexpectedly, donor pigment cells in all the grafts soon selectively proliferated close to areas of greatest wound healing, forming a dense black tracery, especially at the outer rim of the grafts. These lesions slowly grew radially within the grafts, producing irregular greyish patches. Local vertical thickenings then appeared and developed into small melanomas, which soon ulcerated through the epidermis. The tumors rapidly enlarged and became deeply invasive. Discrete black nevi also arose, with many becoming larger and distinctly blue, but those not near areas of pronounced wound healing did not progress to malignancy. In this first series, malignant melanoma resulted in all the grafts from the more susceptible of two donor lines and in some grafts from the other line. Distant metastases occurred in some cases from each line. Most tumors were hypomelanotic and heterogeneous, with lobes or areas differing in melanization. The results strongly suggest that growth factors and cytokines--known to be produced in wound repair--are triggering the growth and malignant conversion of these genetically susceptible melanocytes and that in the graft situation we are merely witnessing a caricature--a usefully exaggerated manifestation of the true events underlying the genesis of melanomas. The striking resemblance to the human malignancy, the genetic uniformity and different susceptibilities of the transgenic lines, and the experimental possibilities in the grafted mice all make them an excellent model of the disease. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5
Mintz, B; Silvers, W K
Genetic marking of sex using a W chromosome-linked transgene.
Many species belonging to the order Lepidoptera are major pests in agriculture and arboriculture. The sterile insect technique (SIT) is an eco-friendly and highly efficient genetically targeted pest management approach. In many cases, it is preferable to release only sterile males in an SIT program, and efficient sexing strategies are crucial to the successful large-scale implementation of SIT. In the present study, we established 160 transgenic silkworm (Bombyx mori) lines to test the possibility of genetic sexing using a W chromosome-linked transgene, which is thought to be the best sexing strategy for lepidopteran species. One transgenic line with a female-specific expression pattern of reporter gene was obtained. The expression level of the W-linked transgene was comparable with autosomal insertions and was stable for 17 continuous generations. Molecular characterization showed this line contained a single copy of the reporter gene on the W chromosome, and the integration site was TTAG in contig W-BAC-522N19-C9. The feasibility of using a W chromosome-linked transgene demonstrated here and the possible improvements discussed will provide valuable information for other lepidopteran pests. The novel W chromosome-linked transgenic line established in this study will serve as an important resource for fundamental research with the silkworm B. mori. PMID:24036279
Ma, Sanyuan; Wang, Xiaogang; Fei, Jitao; Liu, Yuanyuan; Duan, Jianping; Wang, Feng; Xu, Hanfu; Zhao, Ping; Xia, Qingyou
Epigenetic changes of lentiviral transgenes in porcine stem cells derived from embryonic origin.
Because of the physiological and immunological similarities that exist between pigs and humans, porcine pluripotent cell lines have been identified as important candidates for preliminary studies on human disease as well as a source for generating transgenic animals. Therefore, the establishment and characterization of porcine embryonic stem cells (pESCs), along with the generation of stable transgenic cell lines, is essential. In this study, we attempted to efficiently introduce transgenes into Epiblast stem cell (EpiSC)-like pESCs. Consequently, a pluripotent cell line could be derived from a porcine-hatched blastocyst. Enhanced green fluorescent protein (EGFP) was successfully introduced into the cells via lentiviral vectors under various multiplicities of infection, with pluripotency and differentiation potential unaffected after transfection. However, EGFP expression gradually declined during extended culture. This silencing effect was recovered by in vitro differentiation and treatment with 5-azadeoxycytidine. This phenomenon was related to DNA methylation as determined by bisulfite sequencing. In conclusion, we were able to successfully derive EpiSC-like pESCs and introduce transgenes into these cells using lentiviral vectors. This cell line could potentially be used as a donor cell source for transgenic pigs and may be a useful tool for studies involving EpiSC-like pESCs as well as aid in the understanding of the epigenetic regulation of transgenes. PMID:23977247
Choi, Kwang-Hwan; Park, Jin-Kyu; Kim, Hye-Sun; Uh, Kyung-Jun; Son, Dong-Chan; Lee, Chang-Kyu
Transgenics of an elite indica rice variety Pusa Basmati 1 harbouring the codA gene are highly tolerant to salt stress
Transgenic lines of indica rice were generated by Agrobacterium-mediated transformation with the choline oxidase (codA) gene from Arthrobacter globiformis. Choline oxidase catalyses conversion of choline to glycine betaine. Glycine betaine is known to provide tolerance against a variety of stresses. Molecular analyses of seven independent transgenic lines as performed by Southern, Northern and Western hybridization revealed integration and expression of
A. Mohanty; H. Kathuria; A. Ferjani; A. Sakamoto; P. Mohanty; N. Murata; A. K. Tyagi
Gurmarin sensitivity of sweet taste responses is associated with co-expression patterns of T1r2, T1r3, and gustducin.
Gurmarin (Gur) is a peptide that selectively suppresses sweet taste responses in rodents. The inhibitory effect of Gur differs among tongue regions and mouse strains. Recent studies demonstrated that co-expression levels of genes controlling sweet receptors (T1r2/T1r3 heterodimer) versus Galpha-protein, gustducin, are much lower in Gur-insensitive posterior circumvallate papillae than in Gur-sensitive anterior fungiform papillae. Here, we investigated the potential link of Gur-sensitivity with the co-expression for T1r2/T1r3 receptors and gustducin by comparing those of taste tissues of Gur-sensitive (B6, dpa congenic strains) and Gur-weakly-sensitive (BALB) strains. The results indicated that co-expression ratios among T1r2, T1r3, and gustducin in the fungiform papillae were significantly lower in Gur-weakly-sensitive BALB mice than in Gur-sensitive B6 and dpa congenic mice. This linkage between Gur-sensitivity and co-expression for T1r2/T1r3 receptors versus gustducin suggests that gustducin may be a key molecule involved in the pathway for Gur-sensitive sweet responses. PMID:18174025
Shigemura, Noriatsu; Nakao, Kazuko; Yasuo, Toshiaki; Murata, Yoshihiro; Yasumatsu, Keiko; Nakashima, Akihiko; Katsukawa, Hideo; Sako, Noritaka; Ninomiya, Yuzo
Biolistic-mediated genetic transformation of cowpea (Vigna unguiculata) and stable Mendelian inheritance of transgenes.
We describe a novel system of exploiting the biolistic process to generate stable transgenic cowpea (Vigna unguiculata) plants. The system is based on combining the use of the herbicide imazapyr to select transformed meristematic cells after physical introduction of the mutated ahas gene (coding for a mutated acetohydroxyacid synthase, under control of the ahas 5' regulatory sequence) and a simple tissue culture protocol. The gus gene (under control of the act2 promoter) was used as a reporter gene. The transformation frequency (defined as the total number of putative transgenic plants divided by the total number of embryonic axes bombarded) was 0.90%. Southern analyses showed the presence of both ahas and gus expression cassettes in all primary transgenic plants, and demonstrated one to three integrated copies of the transgenes into the genome. The progenies (first and second generations) of all self-fertilized transgenic lines revealed the presence of the transgenes (gus and ahas) co-segregated in a Mendelian fashion. Western blot analysis revealed that the GUS protein expressed in the transgenic plants had the same mass and isoelectric point as the bacterial native protein. This is the first report of biolistic-mediated cowpea transformation in which fertile transgenic plants transferred the foreign genes to next generations following Mendelian laws. PMID:18587583
Ivo, Nayche L; Nascimento, Cristina P; Vieira, LĒvia S; Campos, Francisco A P; AragŃo, Francisco J L
Overexpression of a mutant form of TGFBI/BIGH3 induces retinal degeneration in transgenic mice
Purpose Despite ubiquitous expression of the keratoepithelin (KE) protein encoded by the transforming growth factor beta induced/beta induced gene human clone 3 (TGFBI/BIGH3) gene, corneal dystrophies are restricted to the cornea, and no other tissues are affected. We investigated the role of TGFBI/BIGH3 in Groenouw corneal dystrophies by generating transgenic mice overexpressing TGFBI/BIGH3 containing the R555W mutation. Methods Transgenic animals expressing the Groenouw mutation of human TGFBI/BIGH3 were generated using lentiviral vectors. The line expressed TGFBI/BIGH3 containing the R555W mutation under the control of the phosphoglycerate kinase (PGK) promoter. Expression of the transgene was monitored by Southern and western blotting and by RT¢PCR. Electroretinogram analysis was performed and four mice were subjected to complete necroscopy. Results Transgene expression was observed in different organs although without specific expression in the cornea. The overall morphology of the transgenic animals was not severely affected by KE overexpression. However, we observed an age-dependent retinal degeneration both functionally and histologically. Female-specific follicular hyperplasia in the spleen and increased levels of lipofuscin in the adrenal gland were also seen in transgenic animals. Conclusions Cellular degeneration in the retina of transgenic animals suggest that perturbation of the transforming growth factor beta (TGF?) family regulation may affect photoreceptor survival and may induce possible accelerated aging in several tissues. No corneal phenotype could be observed, probably due to the lack of transgene expression in this tissue.
Maurer, Fabienne; Elkochairi, Ilhem; Lepore, Mario G.; Arsenijevic, Yvan; Pedrazzini, Thierry; Munier, Francis L.; Schorderet, Daniel F.
Progressive 35S promoter methylation increases rapidly during vegetative development in transgenic Nicotiana attenuata plants
Background Genetically modified plants are widely used in agriculture and increasingly in ecological research to enable the selective manipulation of plant traits in the field. Despite their broad usage, many aspects of unwanted transgene silencing throughout plant development are still poorly understood. A transgene can be epigenetically silenced by a process called RNA directed DNA methylation (RdDM), which can be seen as a heritable loss of gene expression. The spontaneous nature of transgene silencing has been widely reported, but patterns of acquirement remain still unclear. Results Transgenic wild tobacco plants (Nicotiana attenuata) expressing heterologous genes coding for antimicrobial peptides displayed an erratic and variable occurrence of transgene silencing. We focused on three independently transformed lines (PNA 1.2, PNA 10.1 and ICE 4.4) as they rapidly lost the expression of the resistance marker and down-regulated transgene expression by more than 200 fold after only one plant generation. Bisulfite sequencing indicated hypermethylation within the 35S and NOS promoters of these lines. To shed light on the progress of methylation establishment, we successively sampled leaf tissues from different stages during plant development and found a rapid increase in 35S promoter methylation during vegetative growth (up to 77% absolute increase within 45Ādays of growth). The levels of de novo methylation were inherited by the offspring without any visible discontinuation. A secondary callus regeneration step could interfere with the establishment of gene silencing and we found successfully restored transgene expression in the offspring of several regenerants. Conclusions The unpredictability of the gene silencing process requires a thorough selection and early detection of unstable plant lines. De novo methylation of the transgenes was acquired solely during vegetative development and did not require a generational change for its establishment or enhancement. A secondary callus regeneration step provides a convenient way to rescue transgene expression without causing undesirable morphological effects, which is essential for experiments that use transformed plants in the analysis of ecologically important traits.
Inducible gene manipulations in brain serotonergic neurons of transgenic rats.
The serotonergic (5-HT) system has been implicated in various physiological processes and neuropsychiatric disorders, but in many aspects its role in normal and pathologic brain function is still unclear. One reason for this might be the lack of appropriate animal models which can address the complexity of physiological and pathophysiological 5-HT functioning. In this respect, rats offer many advantages over mice as they have been the animal of choice for sophisticated neurophysiological and behavioral studies. However, only recently technologies for the targeted and tissue specific modification of rat genes - a prerequisite for a detailed study of the 5-HT system - have been successfully developed. Here, we describe a rat transgenic system for inducible gene manipulations in 5-HT neurons. We generated a Cre driver line consisting of a tamoxifen-inducible CreERT2 recombinase under the control of mouse Tph2 regulatory sequences. Tissue-specific serotonergic Cre recombinase expression was detected in four transgenic TPH2-CreERT2 rat founder lines. For functional analysis of Cre-mediated recombination, we used a rat Cre reporter line (CAG-loxP.EGFP), in which EGFP is expressed after Cre-mediated removal of a loxP-flanked lacZ STOP cassette. We show an in-depth characterisation of this rat Cre reporter line and demonstrate its applicability for monitoring Cre-mediated recombination in all major neuronal subpopulations of the rat brain. Upon tamoxifen induction, double transgenic TPH2-CreERT2/CAG-loxP.EGFP rats show selective and efficient EGFP expression in 5-HT neurons. Without tamoxifen administration, EGFP is only expressed in few 5-HT neurons which confirms minimal background recombination. This 5-HT neuron specific CreERT2 line allows Cre-mediated, inducible gene deletion or gene overexpression in transgenic rats which provides new opportunities to decipher the complex functions of the mammalian serotonergic system. PMID:22140568
Weber, Tillmann; Sch÷nig, Kai; Tews, Bj÷rn; Bartsch, Dusan
Expression of human growth hormone in the milk of transgenic rabbits with transgene mapped to the telomere region of chromosome 7q.
The advent of transgenic technology has provided methods for the production of pharmaceuticals by the isolation of these proteins from transgenic animals. The mammary gland has been focused on as a bioreactor, since milk is easily collected from lactating animals and protein production can be expressed at very high levels, including hormones and enzymes. We demonstrate here the expression pattern of recombinant human growth hormone (rhGH) in transgenic rabbits carrying hGH genomic sequences driven by the rat whey acidic protein (WAP) promoter. The transgene was mapped to the q26-27 telomere region of chromosome 7q by fluorescence in situ hybridization (FISH). Nearly 30 % of the F1 generation demonstrated the presence of transgene. The recombinant growth hormone was detected in the milk of the transgenic rabbit females, but not in serum, up to the level of 10 ?g/ml. Ectopic expression of the transgene in the brain, heart, kidney, liver, and salivary gland was not observed, indicating that a short sequence of rat WAP promoter (969 bp) contained essential sequences directing expression exclusively to the mammary gland. The biological activity of recombinant growth hormone was measured by immunoreactivity and the capability to stimulate growth of the hormone-dependent Nb211 cell line. PMID:22898896
Lipinski, Daniel; Zeyland, Joanna; Szalata, Marlena; Plawski, Andrzej; Jarmuz, Malgorzata; Jura, Jacek; Korcz, Aleksandra; Smorag, Zdzislaw; Pienkowski, Marek; Slomski, Ryszard
5-Azacytidine prevents transgene methylation in vivo.
Retroviral sequence can silence transgene expression in vitro and in vivo. We report that this effect can be efficiently prevented by in vivo administration of the demethylating agent 5-azacytidine (aza-C). We engineered the U937 human cell line with a retroviral vector consisting of the thymidine kinase suicide gene (tk), which induces sensitivity to ganciclovir (gcv) and through an IRES sequence, the bacterial beta-galactosidase gene (lacZ) as a marker gene. About 90% of the U937 cells expressed the transgene. By injecting the transduced U937 cells in severe combined immunodeficient disease (SCID) mice, we generated a tumor which, during in vivo treatment with aza-C, maintained the high expression of lacZ and tk genes at the baseline values. LacZ-positive cells in the tumour masses after death was weak (1-2%) in the control group, while in mice treated with aza-C it was maintained at 90%. The delay in tumour onset was significantly longer when animals were treated with both aza-C and gcv (P < 0.0001) compared with animals treated with gcv or with aza-C alone. The prevention of silencing phenomena has important implications for gene therapy, because an efficient transduction associated with appropriate drug therapy, might be a powerful strategy for successful application of gene therapy protocols. PMID:10476232
Di Ianni, M; Terenzi, A; Perruccio, K; Ciurnelli, R; Lucheroni, F; Benedetti, R; Martelli, M F; Tabilio, A
Surgical Treatment of T1-2 Disc Herniation with T1 Radiculopathy: A Case Report with Review of the Literature
The prevalence of intervertebral disc herniation (IDH) of the thoracic spine is rare compared to the cervical or lumbar spine. In particular, IDH of the upper thoracic spine is extremely rare. We report the case of T1-2 IDH and its treatment, with a literature review. A 37-year-old male patient visited our hospital due to radiating pain at the left upper extremity and weakness of grip power. In cervical spine magnetic resonance images, T1-2 disc space showed herniated disc material and compressed T1 root was identified. Laminoforaminotomy was performed with a posterior approach. The radiating pain and weakness of grip power improved immediately after the surgery. Of patients who show radiating pain or numbness at the medial aspect of forearm, or weakness of intrinsic muscle of hand, can be suspected to have T1 radiculopathy. A detailed physical examination and a radiologic evaluation including this area should be required for the T1 radiculopathy.
Son, Eun-Seok; Park, So-Young; Kim, Ki-Tack; Kang, Chul-Hyung; Cho, Seong-Woo
Adjuvant chemotherapy of pT1a and pT1b breast carcinoma: results from the NEMESI study
Background The prognosis of pT1a-pT1b breast cancer (BC) used to be considered very good, with a 10-y RFS of 90%. However, some retrospective studies reported a 10-y RFS of 81%¢86% and suggested benefit from adjuvant systemic therapy. Methods To evaluate the variables that determined the choice of adjuvant chemotherapy and the type of chemotherapy delivered in pT1a-pT1b BC, we analysed the small tumours enrolled in the NEMESI study. Results Out of 1,894 patients with pathological stage I-II BC enrolled in NEMESI, 402 (21.2%) were pT1a-pT1b. Adjuvant chemotherapy was delivered in 127/402 (31.59%). Younger age, grading G3, high proliferative index, ER-negative and HER2-positive status were significantly associated with the decision to administer adjuvant chemotherapy. An anthracycline without taxane regimen was administered in 59.1% of patients, anthracycline with taxane in 24.4%, a CMF-like regimen in 14.2% and taxane in 2.4%. Adjuvant chemotherapy was administered in 88.4% triple-negative and 73.46% HER2-positive pT1a-pT1b BC. Adjuvant trastuzumab was delivered in 30/49 HER2-positive BC (61.2%). Conclusions Adjuvant chemotherapy was delivered in 31.59% T1a-pT1b BC treated at 63 Italian oncological centres from January 2008 to June 2008. The choice to deliver chemotherapy was based on biological prognostic factors. Anthracycline-based chemotherapy was administered in 83.5% patients.
T1R2 and T1R3 subunits are individually unnecessary for normal affective licking responses to polycose: implications for saccharide taste receptors in mice
The T1R2 and T1R3 proteins are expressed in taste receptor cells and form a heterodimer binding with compounds described as sweet by humans. We examined whether Polycose taste might be mediated through this heterodimer by testing T1R2 knockout (KO) and T1R3 KO mice and their wild-type (WT) littermate controls in a series of brief-access taste tests (25-min sessions with 5-s trials). Sucrose, Na-saccharin, and Polycose were each tested for three consecutive sessions with order of presentation varied among subgroups in a Latin-Square manner. Both KO groups displayed blunted licking responses and initiated significantly fewer trials of sucrose and Na-saccharin across a range of concentrations. KO mice tested after Polycose exposure demonstrated some degree of concentration-dependent licking of sucrose, likely attributable to learning related to prior postingestive experience. These results are consistent with prior findings in the literature, implicating the T1R2+3 heterodimer as the principal taste receptor for sweet-tasting ligands, and also provide support for the potential of postingestive experience to influence responding in the KO mice. In contrast, T1R2 KO and T1R3 KO mice displayed concentration-dependent licking responses to Polycose that tracked those of their WT controls and in some cases licked midrange concentrations more; the number of Polycose trials initiated overall did not differ between KO and WT mice. Thus, the T1R2 and T1R3 proteins are individually unnecessary for normal concentration-dependent licking of Polycose to be expressed in a brief-access test. Whether at least one of these T1R protein subunits is necessary for normal Polycose responsiveness remains untested. Alternatively, there may be a novel taste receptor(s) that mediates polysaccharide taste.
Treesukosol, Yada; Blonde, Ginger D.; Spector, Alan C.
Transgenic farm animals: an update.
The first transgenic livestock species were reported in 1985. Since then microinjection of foreign DNA into pronuclei of zygotes has been the method of choice. It is now being replaced by more efficient protocols based on somatic nuclear transfer that also permit targeted genetic modifications. Lentiviral vectors and small interfering ribonucleic acid (siRNA) technology are also becoming important tools for transgenesis. In 2006 the European Medicines Agency (EMEA) gave green light for the commercialistion of the first recombinant protein produced in the milk of transgenic animals. Recombinant antithrombin III will be launched as ATryn for prophylactic treatment of patients with congenital antithrombin deficiency. This important milestone will boost the research activities in farm animal transgenesis. Recent developments in transgenic techniques of farm animals are discussed in this review. PMID:17714630
Niemann, Heiner; Kues, Wilfried A
Comprehensive assessment of milk composition in transgenic cloned cattle.
The development of transgenic cloned animals offers new opportunities for agriculture, biomedicine and environmental science. Expressing recombinant proteins in dairy animals to alter their milk composition is considered beneficial for human health. However, relatively little is known about the expression profile of the proteins in milk derived from transgenic cloned animals. In this study, we compared the proteome and nutrient composition of the colostrum and mature milk from three lines of transgenic cloned (TC) cattle that specifically express human ?-lactalbumin (TC-LA), lactoferrin (TC-LF) or lysozyme (TC-LZ) in the mammary gland with those from cloned non-transgenic (C) and conventionally bred normal animals (N). Protein expression profile identification was performed, 37 proteins were specifically expressed in the TC animals and 70 protein spots that were classified as 22 proteins with significantly altered expression levels in the TC and C groups compared to N group. Assessment of the relationship of the transgene effect and normal variability in the milk protein profiles in each group indicated that the variation in the endogenous protein profiles of the three TC groups was within the limit of natural variability. More than 50 parameters for the colostrum and mature milk were compared between each TC group and the N controls. The data revealed essentially similar profiles for all groups. This comprehensive study demonstrated that in TC cattle the mean values for the measured milk parameters were all within the normal range, suggesting that the expression of a transgene does not affect the composition of milk. PMID:23185411
Zhang, Ran; Guo, Chengdong; Sui, Shunchao; Yu, Tian; Wang, Jianwu; Li, Ning
Amino acids regulate transgene expression in MDCK cells.
Gene expression and cell growth rely on the intracellular concentration of amino acids, which in metazoans depends on extracellular amino acid availability and transmembrane transport. To investigate the impact of extracellular amino acid concentrations on the expression of a concentrative amino acid transporter, we overexpressed the main kidney proximal tubule luminal neutral amino acid transporter B0AT1-collectrin (SLC6A19-TMEM27) in MDCK cell epithelia. Exogenously expressed proteins co-localized at the luminal membrane and mediated neutral amino acid uptake. However, the transgenes were lost over few cell culture passages. In contrast, the expression of a control transgene remained stable. To test whether this loss was due to inappropriately high amino acid uptake, freshly transduced MDCK cell lines were cultivated either with physiological amounts of amino acids or with the high concentration found in standard cell culture media. Expression of exogenous transporters was unaffected by physiological amino acid concentration in the media. Interestingly, mycoplasma infection resulted in a significant increase in transgene expression and correlated with the rapid metabolism of L-arginine. However, L-arginine metabolites were shown to play no role in transgene expression. In contrast, activation of the GCN2 pathway revealed by an increase in eIF2? phosphorylation may trigger transgene derepression. Taken together, high extracellular amino acid concentration provided by cell culture media appears to inhibit the constitutive expression of concentrative amino acid transporters whereas L-arginine depletion by mycoplasma induces the expression of transgenes possibly via stimulation of the GCN2 pathway. PMID:24797296
Torrente, Marta; Guetg, Adriano; Sass, J÷rn Oliver; Arps, Lisa; Ruckstuhl, Lisa; Camargo, Simone M R; Verrey, Franńois
Amino Acids Regulate Transgene Expression in MDCK Cells
Gene expression and cell growth rely on the intracellular concentration of amino acids, which in metazoans depends on extracellular amino acid availability and transmembrane transport. To investigate the impact of extracellular amino acid concentrations on the expression of a concentrative amino acid transporter, we overexpressed the main kidney proximal tubule luminal neutral amino acid transporter B0AT1-collectrin (SLC6A19-TMEM27) in MDCK cell epithelia. Exogenously expressed proteins co-localized at the luminal membrane and mediated neutral amino acid uptake. However, the transgenes were lost over few cell culture passages. In contrast, the expression of a control transgene remained stable. To test whether this loss was due to inappropriately high amino acid uptake, freshly transduced MDCK cell lines were cultivated either with physiological amounts of amino acids or with the high concentration found in standard cell culture media. Expression of exogenous transporters was unaffected by physiological amino acid concentration in the media. Interestingly, mycoplasma infection resulted in a significant increase in transgene expression and correlated with the rapid metabolism of L-arginine. However, L-arginine metabolites were shown to play no role in transgene expression. In contrast, activation of the GCN2 pathway revealed by an increase in eIF2? phosphorylation may trigger transgene derepression. Taken together, high extracellular amino acid concentration provided by cell culture media appears to inhibit the constitutive expression of concentrative amino acid transporters whereas L-arginine depletion by mycoplasma induces the expression of transgenes possibly via stimulation of the GCN2 pathway.
Torrente, Marta; Guetg, Adriano; Sass, Jorn Oliver; Arps, Lisa; Ruckstuhl, Lisa; Camargo, Simone M. R.; Verrey, Francois
Retention during processing and bioaccessibility of ?-carotene in high ?-carotene transgenic cassava root.
Cassava is a root crop that serves as a primary caloric source for many African communities despite its low content of ?-carotene (?C). Carotenoid content of roots from wild type (WT) and three transgenic lines with high ?C were compared after cooking and preparation of nonfermented and fermented flours according to traditional African methods. The various methods of processing all decreased ?C content per gram dry weight regardless of genotype. The greatest loss of ?C occurred during preparation of gari (dry fermentation followed by roasting) from WT and transgenic lines. The quantities of ?C in cooked transgenic cassava root that partitioned into mixed micelles during in vitro digestion and transported into Caco-2 cells were significantly greater than those for identically processed WT root. These results suggest that transgenic high ?C cassava will provide individuals with greater quantities of bioaccessible ?C. PMID:22458891
Failla, Mark L; Chitchumroonchokchai, Chureeporn; Siritunga, Dimuth; De Moura, Fabiana F; Fregene, Martin; Manary, Mark J; Sayre, Richard T
Using transgenic reporters to visualize bone and cartilage signaling during development in vivo
Green fluorescent protein was first used as a marker of protein expression in vivo 18 years ago, heralding the beginning of what became known as the Green Revolution. Since then, there has been an explosion in the number of transgenic lines in existence, and these transgenic tools are now being applied to skeletal research. Advances in transgenesis are also leading to increasing use of new model organisms for studying skeletogenesis. Such new models include the small teleosts zebrafish and medaka, which due to their optical translucency offer imaging possibilities in the live animals. In this review, we will introduce a number of recent advances in genetic engineering and transgenesis and the new genetic tools that are currently being developed. We will provide examples of how zebrafish and medaka transgenic lines are helping us to understand the behavior of skeletal cells in vivo. Finally, we will discuss future prospects for the application of transgenic technology to skeletal research.
Hammond, Chrissy L.; Moro, Enrico
Transgenic Animals for Testing Multidrug Resistance.
National Technical Information Service (NTIS)
Transgenic animals carrying and expressing human MDR1 gene have been produced. These transgenic animals serve as a useful model for testing the efficacy of high dosage chemotherapy and for the development of novel chemotherapeutic agents against cancers. ...
I. Pastan
Transgenic Animals for Monitoring Water Quality.
National Technical Information Service (NTIS)
The present invention provides methods and systems that uses transgenic zebrafish with an easily assessable reporter gene under the control of pollutant-inducible DNA response elements. Transgenic zebrafish, carrying pollution-inducible response elements,...
D. W. Nebert
How To Produce and Characterize Transgenic Plants.
ERIC Educational Resources Information Center
Explains the process of establishing transgenic plants which is a very important tool in plant biology and modern agriculture. Produces transgenic plants with the ability to synthesize opines. (Contains 17 references.) (YDS)
Savka, Michael A.; Wang, Shu-Yi; Wilson, Mark
Prognostic role of substaging in T1G3 transitional cell carcinoma of the urinary bladder
This study was conducted to test a new substaging system in a population of patients with stage T1 bladder cancer (BC) at diagnosis and assess its prognostic role in terms of disease progression and disease-specific survival (DSS). Patients with primary stage T1G3 urothelial carcinoma of the bladder were stratified according to the following models: i) T1a [the tumour does not infiltrate the muscularis mucosae-vascular plexus, (MM-VP)]; T1b (the tumour partially infiltrates the MM-VP); and T1c (the tumour infiltrates and invades the MM-VP). ii) T1m (diameter of tumour infiltrating the lamina propria ?0.5 mm under a high-resolution microscope; and T1e (diameter of tumour infiltrating the lamina propria >0.5 mm). Age, gender, tumour size and multifocality were not found to be of statistical significance. Using the T1a/T1b/T1c system, patients with stage T1a disease exhibited a 5- and 10-year progression rate of 13.3 and 20%, respectively, without reaching statistical significance. Moreover, patients with stage T1a disease exhibited a 5- and 10-year DSS of 93.3 and 73.3%, respectively, which was higher compared to T1b and T1c but not statistically significant. Using the T1m/T1e system, patients with stage T1m disease exhibited a disease progression rate of 8.3 and 16.7% at 5 and 10 years, respectively, which was not statistically significant. Moreover, patients in group T1m presented with DSS rates of 91.7 and 83.3% at 5 and 10 years, respectively, which were higher compared to those in the T1e group (71.4 and 60.7%), although not reaching statistical significance. In conclusion, in our study, neither of the two substaging systems of stage T1 BC reached the prognostic conventional significance level for tumour progression or DSS.
Expression of recombinant human alpha-lactalbumin in the milk of transgenic goats using a hybrid pomoter/enhancer.
To improve nutrient content of goat milk, we describe the construction of a vector (pBLAC) containing a hybrid goat ? -lactoglobulin (BLG) promoter/cytomegalovirus (CMV) enhancer. We also describe the generation of transgenic goats expressing rhLA by somatic cell nuclear transfer (SCNT). Of 334 one-cell stage embryos derived from three transgenic cell lines and 99 embryos derived from non-transgenic (NT) cells surgically transferred to the oviducts of 37 recipients, two recipients delivered two kids (2%) from the non-transfected line and five recipients delivered six kids (1.8%) from transgenic cell lines, three of which died within 2 days. Compared to the NT donor cells, transfection of donor cells does not negatively affect the development of nuclear transfer embryos into viable transgenic offspring. However, the clone efficiency in cell line number 1 was lower than that in numbers 2 and 3, and in the NT lines (0.9% versus 1.9% 2.4% and 2%; P < 0.05). Two transgenic cloned goats expressed rhLA in the milk at 0.1-0.9?mg/mL. The mammary gland-specific expression vector pBLAC with hybrid BLG/CMV can drive the hLA gene to express in vitro and in vivo. These data establish the basis for use of a hybrid promoter/enhancer strategy to produce rhLA transgenic goats. PMID:24527256
Yuan, Yu-Guo; An, Liyou; Yu, Baoli; Song, Shaozheng; Zhou, Feng; Zhang, Liqing; Gu, Yinyin; Yu, Minghui; Cheng, Yong
Approaches to Minimize Variation of Transgene Expression in Plants
Genetic transformation of plants has become a widely used technology that serves multiple purposes in plant biology research. However, considerable variation of transgene expression is often observed within populations of transgenic plants transformed with the same transgene construct. This inter-transformant variation of transgene expression hampers proper evaluation of transgenes and might be most undesirable when high-throughput transgene screening is intended.
Katleen M. J. Butaye; Bruno P. A. Cammue; Stijn L. Delaureand; Miguel F. C. De Bolle
Production of functional transgenic mice by DNA pronuclear microinjection
Successful experiments involving the production of transgenic mice by pronuclear microinjection are currently limited by low efficiency of random transgene integra- tion into the mouse genome. Furthermore, not all transgenic mice express integrated transgenes, or in other words are effective as functional transgenic mice expressing the desired product of the transgene, thus allowing accomplishment of the ultimate experimental goal -
Anna B. Auerbach
Production of transgenic lily plants by Agrobacterium-mediated transformation.
A system for the production of transgenic plants was developed for the Oriental hybrid lily, Lilium cv. Acapulco, by Agrobacterium-mediated genetic transformation. Filament-derived calli were co-cultivated with A. tumefaciens strain EHA101/pIG121Hm, which harbored a binary vector carrying the neomycin phosphotransferase II, hygromycin phosphotransferase, and intron-containing beta-glucuronidase genes in the T-DNA region. Six hygromycin-resistant (Hyg(r)) culture lines were obtained from 200 calli by scratching them with sandpaper prior to inoculation and using NH(4)NO(3)-free medium for co-cultivation and a hygromycin-containing regeneration medium for selection. Hyg(r) culture lines regenerated shoots, which developed into plantlets following transfer to a plant growth regulator-free medium. All of these plantlets were verified to be transgenic by GUS histochemical assay and inverse PCR analysis. PMID:14685763
Hoshi, Y; Kondo, M; Mori, S; Adachi, Y; Nakano, M; Kobayashi, H
Expression of human protoporphyrinogen oxidase in transgenic rice induces both a photodynamic response and oxyfluorfen resistance
A human protoporphyrinogen oxidase (Protox) coding sequence under the control of a ubiquitin promoter was introduced into rice to determine whether transgenic rice overexpressing the human Protox gene exhibits resistance against a peroxidizing herbicide. The transgenic rice lines (H3, H4, H5, H6, H9, and H10) transcribed the human Protox mRNA, whereas hybridizing RNA band was not detected in wild-type rice,
Y. Lee; S. Jung; K. Back
Synergistic activity of chitinases and ?-1,3-glucanases enhances fungal resistance in transgenic tomato plants
SummaryĀĀSimultaneous expression of a tobacco class I chitinase and a class I ?-1,3-glucanase gene in tomato resulted in increased\\u000a fungal resistance, whereas transgenic tomato plants expressing either one of these genes were not protected against fungal\\u000a infection. After infection with Fusarium oxysporum f.sp. lycopersici, a 36% to 58% reduction in disease severity was observed in resistant tomato lines. Two transgenic
Erik Jongedijk; Henk Tigelaar; Jeroen S. C. van Roekel; Sandra A. Bres-Vloemans; Ilma Dekker; Ben J. C. Cornelissen; Leo S. Melchers
Resistance mechanisms in protoporphyrinogen oxidase (PROTOX) inhibitor-resistant transgenic rice
We investigated the mechanism for conferring herbicide resistance in transgenic rice. Plants from Line M4 were resistant to\\u000a PROTOX inhibitors and had yields similar to those from wild-type (WT) rice.Myxococcus xanthus PROTOX mRNA was abundantly expressed in the transgenic leaf tissues, and theM. xanthus PROTOX gene was stably transmitted into the T4 generation. We detected a protein with a predicted
Ha Il Jung; Yong In Kuk
Transgene Expression Is Associated with Copy Number and Cytomegalovirus Promoter Methylation in Transgenic Pigs
Transgenic animals have been used for years to study gene function, produce important proteins, and generate models for the study of human diseases. However, inheritance and expression instability of the transgene in transgenic animals is a major limitation. Copy number and promoter methylation are known to regulate gene expression, but no report has systematically examined their effect on transgene expression. In the study, we generated two transgenic pigs by somatic cell nuclear transfer (SCNT) that express green fluorescent protein (GFP) driven by cytomegalovirus (CMV). Absolute quantitative real-time PCR and bisulfite sequencing were performed to determine transgene copy number and promoter methylation level. The correlation of transgene expression with copy number and promoter methylation was analyzed in individual development, fibroblast cells, various tissues, and offspring of the transgenic pigs. Our results demonstrate that transgene expression is associated with copy number and CMV promoter methylation in transgenic pigs.
Kong, Qingran; Wu, Meiling; Huan, Yanjun; Zhang, Li; Liu, Haiyan; Bou, Gerelchimeg; Luo, Yibo; Mu, Yanshuang; Liu, Zhonghua
Transgenic Tieg Non-Human Animals.
National Technical Information Service (NTIS)
Materials and methods related to a transgenic non-human animal (e.g., a transgenic non-human mammal) whose genome comprises a disrupted TIEG allele are provided. Methods for making such transgenic non-human animals, and using them to identify and characte...
M. Subramaniam, M. J. Velasquez, N. M. Rajmannan, T. C. Spelsberg
Conspiracy of silence among repeated transgenes
Summary Transgenic experiments in vertebrates often involve the insertion of tandem multiple-copy arrays at single sites. For many transgenes, expression is unpredict- able from site to site, a phenomenon usually attributed to a repressive environment caused by nearby sequences. However, an alternative explanation comes from evidence that transgene repeat arrays in flies condense into heterochromatin, suggesting that low levels of
Steven Henikoff
Butyrate Transcriptionally Enhances Peptide Transporter PepT1 Expression and Activity
Background PepT1, an intestinal epithelial apical di/tripeptide transporter, is normally expressed in the small intestine and induced in colon during chronic inflammation. This study aimed at investigating PepT1 regulation by butyrate, a short-chain fatty acid produced by commensal bacteria and accumulated inside inflamed colonocyte. Results We found that butyrate treatment of human intestinal epithelial Caco2-BBE cells increased human PepT1 (hPepT1) promoter activity in a dose- and time-dependent manner, with maximal activity observed in cells treated with 5 mM butyrate for 24 h. Under this condition, hPepT1 promoter activity, mRNA and protein expression levels were increased as assessed by luciferase assay, real-time RT-PCR and Western blot, respectively. hPepT1 transport activity was accordingly increased by ?2.5-fold. Butyrate did not alter hPepT1 mRNA half-life indicating that butyrate acts at the transcriptional level. Molecular analyses revealed that Cdx2 is the most important transcription factor for butyrate-induced increase of hPepT1 expression and activity in Caco2-BBE cells. Butyrate-activated Cdx2 binding to hPepT1 promoter was confirmed by gel shift and chromatin immunoprecipitation. Moreover, Caco2-BBE cells overexpressing Cdx2 exhibited greater hPepT1 expression level than wild-type cells. Finally, treatment of mice with 5 mM butyrate added to drinking water for 24 h increased colonic PepT1 mRNA and protein expression levels, as well as enhanced PepT1 transport activity in colonic apical membranes vesicles. Conclusions Collectively, our results demonstrate that butyrate increases PepT1 expression and activity in colonic epithelial cells, which provides a new understanding of PepT1 regulation during chronic inflammation.
Dalmasso, Guillaume; Nguyen, Hang Thi Thu; Yan, Yutao; Charrier-Hisamuddin, Laetitia; Sitaraman, Shanthi V.; Merlin, Didier
Field performance of transgenic citrus trees: Assessment of the long-term expression of uidA and nptII transgenes and its impact on relevant agronomic and phenotypic characteristics
Background The future of genetic transformation as a tool for the improvement of fruit trees depends on the development of proper systems for the assessment of unintended effects in field-grown GM lines. In this study, we used eight transgenic lines of two different citrus types (sweet orange and citrange) transformed with the marker genes ?-glucuronidase (uidA) and neomycin phosphotransferase II (nptII) as model systems to study for the first time in citrus the long-term stability of transgene expression and whether transgene-derived pleiotropic effects occur with regard to the morphology, development and fruit quality of orchard-grown GM citrus trees. Results The stability of the integration and expression of the transgenes was confirmed in 7-year-old, orchard-grown transgenic lines by Southern blot analysis and enzymatic assays (GUS and ELISA NPTII), respectively. Little seasonal variation was detected in the expression levels between plants of the same transgenic line in different organs and over the 3?years of analysis, confirming the absence of rearrangements and/or silencing of the transgenes after transferring the plants to field conditions. Comparisons between the GM citrus lines with their non-GM counterparts across the study years showed that the expression of these transgenes did not cause alterations of the main phenotypic and agronomic plant and fruit characteristics. However, when comparisons were performed between diploid and tetraploid transgenic citrange trees and/or between juvenile and mature transgenic sweet orange trees, significant and consistent differences were detected, indicating that factors other than their transgenic nature induced a much higher phenotypic variability. Conclusions Our results indicate that transgene expression in GM citrus remains stable during long-term agricultural cultivation, without causing unexpected effects on crop characteristics. This study also shows that the transgenic citrus trees expressing the selectable marker genes that are most commonly used in citrus transformation were substantially equivalent to the non-transformed controls with regard to their overall agronomic performance, as based on the use of robust and powerful assessment techniques. Therefore, future studies of the possible pleiotropic effects induced by the integration and expression of transgenes in field-grown GM citrus may focus on the newly inserted trait(s) of biotechnological interest.
B islet cells of pancreas are the site of expression of the human insulin gene in transgenic mice
Transgenic mouse lines carrying the human insulin gene were previously shown to express it in pancreas but not in other tissues. The present study reports evidence that the expression of the transgene is restricted to a single category of cells. Immunofluorescence staining of frozen pancreas sections showed that the human C-peptide was present in pancreatic islets only, and more precisely in the B cells of the islets. Human insulin transcripts were initiated correctly in mouse pancreas at the same site as in human pancreas. Three different transgenic lines with different insertion sites and various copy numbers of the human insulin transgene had the same high levels of the transgene transcripts corresponding to a well-balanced contribution in insulin gene expression.
Bucchini, D.; Desbois, P.; Pictet, R.; Jami, J. (Univ. Paris 7 (France)); Madsen, O. (Hagedorn Research Lab., Gentofte (Denmark))
Inhibition of HIV-1 Transcription and Replication by a Newly Identified Cyclin T1 Splice Variant*
A variety of cellular factors participates in the HIV-1 life cycle. Among them is the well characterized cyclin T1 (CYCT1). CycT1 binds to cyclin-dependent kinase 9 (CDK9) and forms the positive transcription elongation factor-b (P-TEFb). P-TEFb is then recruited by HIV-1 TAT to the HIV-1 long terminal repeat (LTR) promoter and subsequently leads to phosphorylation of the C-terminal domain of RNA polymerase II (pol II), enhanced processivity of pol II, and transcription of a full-length HIV-1 RNA. In this study, we report the identification of a new CYCT1 splice variant, designated as CYCT1b, and accordingly we renamed CYCT1 as CYCT1a. CYCT1b was detected in several cell lines, including primary human CD4 T cells, and its expression was subject to cell cycle regulation. Similar to CYCT1a, CYCT1b was primarily localized in the nucleus. CYCT1b expression was found to be inversely correlated with HIV-1 gene expression and replication. This inverse correlation appeared to involve TAT transactivation, CDK9 binding, and subsequent recruitment of P-TEFb to the HIV-1 LTR promoter and pol II C-terminal domain phosphorylation. In agreement with these findings, CYCT1b expression led to direct inhibition of TAT-transactivated transcription of the HIV-1 LTR promoter. Taken together, these results show that the newly identified CYCT1b splice variant inhibits HIV-1 transcription and may provide new clues for the development of anti-HIV strategies.
Gao, Guozhen; Wu, Xiaoyun; Zhou, Jieqiong; He, Mingfeng; He, Johnny J.; Guo, Deyin
Divergent phenotypes in mutant TDP-43 transgenic mice highlight potential confounds in TDP-43 transgenic modeling.
The majority of cases of frontotemporal lobar degeneration and amyotrophic lateral sclerosis are pathologically defined by the cleavage, cytoplasmic redistribution and aggregation of TAR DNA binding protein of 43 kDa (TDP-43). To examine the contribution of these potentially toxic mechanisms in vivo, we generated transgenic mice expressing human TDP-43 containing the familial amyotrophic lateral sclerosis-linked M337V mutation and identified two lines that developed neurological phenotypes of differing severity and progression. The first developed a rapid cortical neurodegenerative phenotype in the early postnatal period, characterized by fragmentation of TDP-43 and loss of endogenous murine Tdp-43, but entirely lacking aggregates of ubiquitin or TDP-43. A second, low expressing line was aged to 25 months without a severe neurodegenerative phenotype, despite a 30% loss of mouse Tdp-43 and accumulation of lower molecular weight TDP-43 species. Furthermore, TDP-43 fragments generated during neurodegeneration were not C-terminal, but rather were derived from a central portion of human TDP-43. Thus we find that aggregation is not required for cell loss, loss of murine Tdp-43 is not necessarily sufficient in order to develop a severe neurodegenerative phenotype and lower molecular weight TDP-43 positive species in mouse models should not be inherently assumed to be representative of human disease. Our findings are significant for the interpretation of other transgenic studies of TDP-43 proteinopathy. PMID:24466128
D'Alton, Simon; Altshuler, Marcelle; Cannon, Ashley; Dickson, Dennis W; Petrucelli, Leonard; Lewis, Jada
Transgenic Manipulation of the Metabolism of Polyamines in Poplar Cells1
The metabolism of polyamines (putrescine, spermidine, and spermine) has become the target of genetic manipulation because of their significance in plant development and possibly stress tolerance. We studied the polyamine metabolism in non-transgenic (NT) and transgenic cells of poplar (Populus nigra ū maximowiczii) expressing a mouse Orn decarboxylase (odc) cDNA. The transgenic cells showed elevated levels of mouse ODC enzyme activity, severalfold higher amounts of putrescine, a small increase in spermidine, and a small reduction in spermine as compared with NT cells. The conversion of labeled ornithine (Orn) into putrescine was significantly higher in the transgenic than the NT cells. Whereas exogenously supplied Orn caused an increase in cellular putrescine in both cell lines, arginine at high concentrations was inhibitory to putrescine accumulation. The addition of urea and glutamine had no effect on polyamines in either of the cell lines. Inhibition of glutamine synthetase by methionine sulfoximine led to a substantial reduction in putrescine and spermidine in both cell lines. The results show that: (a) Transgenic expression of a heterologous odc gene can be used to modulate putrescine metabolism in plant cells, (b) accumulation of putrescine in high amounts does not affect the native arginine decarboxylase activity, (c) Orn biosynthesis occurs primarily from glutamine/glutamate and not from catabolic breakdown of arginine, (d) Orn biosynthesis may become a limiting factor for putrescine production in the odc transgenic cells, and (e) assimilation of nitrogen into glutamine keeps pace with an increased demand for its use for putrescine production.
Bhatnagar, Pratiksha; Glasheen, Bernadette M.; Bains, Suneet K.; Long, Stephanie L.; Minocha, Rakesh; Walter, Christian; Minocha, Subhash C.
Application of Schr÷dinger equation to study the tunnelling dynamics of proton transfer in the hydrogen bond of 2,5-dinitrobenzoic acid: proton T1 T1rho, and deuteron T1 relaxation methods.
Temperature measurements of proton T1 (24.7 MHz), deuteron (deuterated hydroxyl group) T1 (55.2 MHz), and proton T1(rho) (B1 = 9 G) spin-lattice relaxation times of 2,5-dinitrobenzoic acid have been performed. An analysis of present experimental data together with previously published proton T1 (55.2 MHz) data has revealed the following molecular motions: proton/deuteron transfer in the hydrogen bond and two-site hopping of the whole dimer. It is shown that the proton-transfer dynamics are characterized by two correlation times tau(ov) and tau(tu), describing two fundamentally different motional processes, namely, thermally activated jumps over the barrier and tunneling through the barrier. The temperature dependence of 1/tau(tu) is the solution of Schr÷dinger's equation, which also yields the temperature T(tun), where begins the tunnel pathway for proton transfer. A new equation for the spectral density function of complex motion consisting of the three motions is derived. The third motion (two-site hopping of the whole dimer characterized by tau(lib) correlation time) is responsible for a proton T1(rho) minimum in high temperatures, just below the melting point. Such a minimum is not reached by T1 temperature dependencies. The minimum of T1(rho) assigned to the classical hopping of a hydrogen-bonded proton occurs in the same low-temperature regime in which the flattening of the temperature dependencies of T1 points to the dominance of incoherent tunneling. This experimental fact denies the known theories predicting the intermediate temperature regime where a smooth transition between classical and quantum tunneling dynamics is expected. The fit of the derived theoretical equations to the experimental data T1(rho) and T1 is satisfactory. The correlation times obtained for deuterons indicate deuteron-transfer dynamics much slower than proton-transfer dynamics. It is concluded that the classical proton transfer takes place over the whole temperature regime, while the incoherent tunneling occurs below 46.5 (hydrogen) or 87.2 K (deuterium) only. PMID:17263515
Latanowicz, L; Medycki, W
Transgenic Nicotiana tabacum plants expressing a fungal copper transporter gene show enhanced acquisition of copper.
The diets of two-thirds of the world's population are deficient in one or more essential elements and one of the approaches to enhance the levels of mineral elements in food crops is by developing plants with ability to accumulate them in edible parts. Besides conventional methods, transgenic technology can be used for enhancing metal acquisition in plants. Copper is an essential element, which is often deficient in human diet. With the objective of developing plants with improved copper acquisition, a high-affinity copper transporter gene (tcu-1) was cloned from fungus Neurospora crassa and introduced into a model plant (Nicotiana tabacum). Integration of the transgene was confirmed by Southern blot hybridization. Transgenic tobacco plants (T(0) and T(1)) expressing tcu-1, when grown in hydroponic medium spiked with different concentrations of copper, showed higher acquisition of copper (up to 3.1 times) compared with control plants. Transgenic plants grown in soil spiked with copper could also take up more copper compared with wild-type plants. Supplementation of other divalent cations such as Cd(2+) and Zn(2+) did not alter uptake of Cu by transgenic plants. The present study has shown that expression of a heterologous copper transporter in tobacco could enhance acquisition of copper. PMID:21671073
Singh, Sudhir; Korripally, Premsagar; Vancheeswaran, Ramachandran; Eapen, Susan
Dynamic Smad-mediated BMP signaling revealed through transgenic zebrafish
BMP signaling is fundamental to development, injury response, and homeostasis. We have developed transgenic zebrafish that report Smad-mediated BMP signaling in embryos and adults. These lines express either eGFP, destabilized eGFP, or destabilized KO2 under the well-characterized æBMP Response ElementÆ (BRE). These fluorescent proteins were found to be expressed dynamically in regions of known BMP signaling including the developing tailbud, hematopoietic lineage, dorsal eye, brain structures, heart, jaw, fins, and somites, as well as other tissues. Responsiveness to changes in BMP signaling was confirmed by observing fluorescence after activation in an hsp70:bmp2b transgenic background or by inhibition in an hsp70:nog3 background. We further demonstrated faithful reportage by the BRE transgenic lines following chemical repression of BMP signaling using an inhibitor of BMP receptor activity, dorsomorphin. Overall, these lines will serve as valuable tools to explore the mechanisms and regulation of BMP signal during embryogenesis, in tissue maintenance, and during disease.
Collery, Ross F.; Link, Brian A.
The a"MAZE"ing world of lung specific transgenic mice
The purpose of this review is to give a comprehensive overview of transgenic mouse lines suitable for studying gene function and cellular lineage relationships in lung development, homeostasis, injury and repair. Many of the mouse strains reviewed in this article have been widely shared within the lung research community and new strains are continuously being developed. There are many useful transgenic lines that work to target subsets of lung cells, but it remains a challenge for investigators to select the correct transgenic modules for their experiment. This review covers both the tetracycline and tamoxifen inducible systems and will primarily focus on conditional lines that target the epithelial cells. We point out the limitations of each strain so investigators can choose the system that will work best for their scientific question. Current mesenchymal and endothelial lines are limited by the fact that they are not lung specific. These lines will be summarized in a brief overview. In addition, useful transgenic reporter mice for studying lineage relationships, promoter activity and signaling pathways will complete our lung specific conditional transgenic mouse-shopping list.
Rawlins, Emma L.; Perl, Anne-Karina
The a"MAZE"ing world of lung-specific transgenic mice.
The purpose of this review is to give a comprehensive overview of transgenic mouse lines suitable for studying gene function and cellular lineage relationships in lung development, homeostasis, injury, and repair. Many of the mouse strains reviewed in this Perspective have been widely shared within the lung research community, and new strains are continuously being developed. There are many transgenic lines that target subsets of lung cells, but it remains a challenge for investigators to select the correct transgenic modules for their experiment. This review covers the tetracycline- and tamoxifen-inducible systems and focuses on conditional lines that target the epithelial cells. We point out the limitations of each strain so investigators can choose the system that will work best for their scientific question. Current mesenchymal and endothelial lines are limited by the fact that they are not lung specific. These lines are summarized in a brief overview. In addition, useful transgenic reporter mice for studying lineage relationships, promoter activity, and signaling pathways will complete our lung-specific conditional transgenic mouse shopping list. PMID:22180870
Rawlins, Emma L; Perl, Anne-Karina
Transgene-like animal models using intronic microRNAs.
Transgenic animal models are valuable tools for testing gene functions and drug mechanisms in vivo. They are also the best similitude of a human body for etiological and pathological research of diseases. All pharmaceutically developed drugs must be proven safe and effective in animals before approval by the Food and Drug Administration to be used in clinical trials. To this end, the transgenic animal models of human diseases serve as a front line for drug evaluation. However, there is currently no transgenic animal model for microRNA (miRNA) research. miRNAs, small single-stranded regulatory RNAs capable of silencing intracellular gene transcripts that contain either complete or partial complementarity to the miRNAs, are useful for the design and development of new therapies against cancer polymorphism and viral mutation. Recently, varieties of natural miRNAs have been found to be derived from hairpin-like RNA precursors in almost all eukaryotes, including yeast (Schizosaccharomyces pombe), plant (Arabidopsis), nematode (Caenorhabditis elegans), fly (Drosophila melanogaster), fish, mouse, and human, involving intracellular defense against viral infections and regulation of certain gene expressions during development. To facilitate the miRNA research in vivo, we have developed a state-of-the-art transgenic strategy for silencing specific genes in zebrafish, chicken, and mouse, using intronic miRNAs. By insertion of a hairpin-like pre-miRNA structure into the intron region of a gene, we have found that mature miRNAs were successfully transcribed by RNA polymerase (Pol)-II, coexpressed with the encoding gene transcript, and excised out of the encoding gene transcript by natural RNA splicing and processing mechanisms. In conjunction with retroviral transfection systems, the hairpin-like pre-miRNA construct was further inserted into the intron of a cellular gene for tissue-specific expression regulated by the gene promoter. Because the retroviral vectors were randomly integrated into the genome of its host cell, the most effective transgenic animal can be selected and propagated to be a stable transgenic line for future research. Here, we have shown for the first time that transgene-like animal models were generated using the intronic miRNA-expressing system described previously, which has been proven to be useful for both miRNA research and in vivo evaluation of miRNA-associated target genes. PMID:16957386
Lin, Shi-Lung; Chang, Shin-Ju E; Ying, Shao-Yao
Cytosine methylation of an Sp1 site contributes to organ-specific and cell-specific regulation of expression of the lung epithelial gene t1alpha.
Several recent observations have suggested that cytosine methylation has a role in the in vivo transcriptional regulation of cell-specific genes in normal cells. We hypothesized that methylation regulates T1alpha, a gene expressed primarily in lung in adult rodents. In fetuses T1alpha is expressed in several organs, including the entire nervous system, but during development its expression is progressively restricted to lung alveolar type I epithelial cells, some osteoblasts and choroid plexus. Here we report that T1alpha is methylated at a key Sp1 site in the proximal promoter in cells and organs, including brain, where no gene expression is detectable. Conversely, in T1alpha-expressing cells, these sites are not methylated. In embryonic brain T1alpha is unmethylated and expressed; in adult brain the gene is methylated and not expressed. In lung epithelial cell lines, methylation of the T1alpha promoter in vitro decreases expression by approx. 50% (the maximum suppression being 100%). Analysis of mutated promoter constructs indicates that a single Sp1 site in the proximal promoter provides all or most of the methylation-sensitive gene silencing. We conclude that, in addition to regulation by transcription factors, cytosine methylation has a role in the complex expression patterns of this gene in intact animals and primary cells.
Cao, Y X; Jean, J C; Williams, M C
Truncated Forms of Pax6 Disrupt Lens Morphology in Transgenic Mice
RESULTS. Two lines of PD 1 HD mice and three lines of PD mice were generated, all of which exhibit posterior nuclear and\\/or cortical cataracts of variable severity. The lenses from mice transgenic for either Pax-6 truncation are smaller and more hydrated than normal. Morphologically, the mice expressing the PD 1 HD of Pax-6 have swollen lens fibers with attenuated
Melinda K. Duncan; Ales Cvekl; Xuan Li; Joram Piatigorsky
Hypertriglyceridemia as a Result of Human Apo CIII Gene Expression in Transgenic Mice
Primary and secondary hypertriglyceridemia is common in the general population, but the biochemical basis for this disease is largely unknown. With the use of transgenic technology, two lines of mice were created that express the human apolipoprotein CIII gene. One of these mouse lines with 100 copies of the gene was found to express large amounts of the protein and
Yasushi Ito; Neal Azrolan; Anita O'Connell; Annemarie Walsh; Jan L. Breslow
Spi-1/PU.1 transgenic mice develop multistep erythroleukemias.
Insertional mutagenesis of the spi-1 gene is associated with the emergence of malignant proerythroblasts during Friend virus-induced acute erythroleukemia. To determine the role of spi-1/PU.1 in the genesis of leukemia, we generated spi-1 transgenic mice. In one founder line the transgene was overexpressed as an unexpected-size transcript in various mouse tissues. Homozygous transgenic animals gave rise to live-born offspring, but 50% of the animals developed a multistep erythroleukemia within 1.5 to 6 months of birth whereas the remainder survived without evidence of disease. At the onset of the disease, mice became severely anemic. Their hematopoietic tissues were massively invaded with nontumorigenic proerythroblasts that express a high level of Spi-1 protein. These transgenic proerythroblasts are partially blocked in differentiation and strictly dependent on erythropoietin for their proliferation both in vivo and in vitro. A complete but transient regression of the disease was observed after erythrocyte transfusion, suggesting that the constitutive expression of spi-1 is related to the block of the differentiation of erythroid precursors. At relapse, erythropoietin-independent malignant proerythroblasts arose. Growth factor autonomy could be partially explained by the autocrine secretion of erythropoietin; however, other genetic events appear to be necessary to confer the full malignant phenotype. These results reveal that overexpression of spi-1 is essential for malignant erythropoiesis and does not alter other hematopoietic lineages.
Moreau-Gachelin, F; Wendling, F; Molina, T; Denis, N; Titeux, M; Grimber, G; Briand, P; Vainchenker, W; Tavitian, A
Mechanism of random integration of foreign DNA in transgenic mice.
Little is known about how foreign DNA is randomly integrated into chromosomes in transgenic animals. In the current study, the insertion sites of 36 transgenic mice were mapped by thermal asymmetric interlaced PCR, and 38 junction sequences were obtained from 30 samples. Analysis of the 38 sequences revealed that 44.7Ā% of integration events occurred within host gene regions, including 13.2Ā% (5/38) in exonic regions and 31.6Ā% (12/38) in intronic regions. The results also revealed that all non-end side integrations of foreign DNA were mediated by short sequence homologies (microhomologies) and that the end side integrations occurred in the presence or absence of microhomologies. In addition, microhomology-mediated mechanisms were also confirmed in four transgenic Arabidopsis thaliana lines. The results indicate that foreign DNA is easily integrated into host gene regions. These results also suggest that the integration of both ends of foreign DNA follows the above-mentioned mechanism in many transgenic/transformed organisms. PMID:23483296
Yan, Bo-Wen; Zhao, Yao-Feng; Cao, Wen-Guang; Li, Ning; Gou, Ke-Mian
In vivo mutation analysis using the ?X174 transgenic mouse and comparisons with other transgenes and endogenous genes.
The ?X174 transgenic mouse was first developed as an in vivo Ames test, detecting base pair substitution (bps) at a single bp in a reversion assay. A forward mutational assay was also developed, which is a gain of function assay that also detects bps exclusively. Later work with both assays focused on establishing that a mutation was fixed in vivo using single-burst analysis: determining the number of mutant progeny virus from an electroporated cell by dividing the culture into aliquots before scoring mutants. We review results obtained from single-burst analysis, including testing the hypothesis that high mutant frequencies (MFs) of G:C to A:T mutation recovered by transgenic targets include significant numbers of unrepaired G:T mismatches. Comparison between the ?X174 and lacI transgenes in mouse spleen indicates that the spontaneous bps mutation frequency per nucleotide (mf(n)) is not significantly lower for ?X174 than for lacI; the response to ENU is also comparable. For the lacI transgene, the spontaneous bps mf(n) is highly age-dependent up to 12 weeks of age and the linear trend extrapolates at conception to a frequency close to the human bps mf(n) per generation of 1.7 ū 10(-8). Unexpectedly, we found that the lacI somatic (spleen) bps mf(n) per cell division at early ages was estimated to be the same as for the human germ-line. The bps mf(n) in bone marrow for the gpt transgene is comparable to spleen for the lacI and ?X174 transgenes. We conclude that the G:C to A:T transition is characteristic of spontaneous in vivo mutation and that the MFs measured in these transgenes at early ages reflect the expected accumulation of in vivo mutation typical of endogenous mammalian mutation rates. However, spontaneous and induced mf(n)s per nucleotide for the cII gene in spleen are 5-10 times higher than for these other transgenes. PMID:20637298
Valentine, Carrie R; Delongchamp, Robert R; Pearce, Mason G; Rainey, Heather F; Dobrovolsky, Vasily N; Malling, Heinrich V; Heflich, Robert H
Characterization of FLOWERING LOCUS T1 (FT1) Gene in Brachypodium and Wheat.
The phase transition from vegetative to reproductive growth is a critical event in the life cycle of flowering plants. FLOWERING LOCUS T (FT) plays a central role in the regulation of this transition by integrating signals from multiple flowering pathways in the leaves and transmitting them to the shoot apical meristem. In this study, we characterized FT homologs in the temperate grasses Brachypodium distachyon and polyploid wheat using transgenic and mutant approaches. Downregulation of FT1 by RNAi was associated with a significant downregulation of the FT-like genes FT2 and FT4 in Brachypodium and FT2 and FT5 in wheat. In a transgenic wheat line carrying a highly-expressed FT1 allele, FT2 and FT3 were upregulated under both long and short days. Overexpression of FT1 caused extremely early flowering during shoot regeneration in both Brachypodium and hexaploid wheat, and resulted in insufficient vegetative tissue to support the production of viable seeds. Downregulation of FT1 transcripts by RNA interference (RNAi) resulted in non-flowering Brachypodium plants and late flowering plants (2-4 weeks delay) in wheat. A similar delay in heading time was observed in tetraploid wheat plants carrying mutations for both FT-A1 and FT-B1. Plants homozygous only for mutations in FT-B1 flowered later than plants homozygous only for mutations in FT-A1, which corresponded with higher transcript levels of FT-B1 relative to FT-A1 in the early stages of development. Taken together, our data indicate that FT1 plays a critical role in the regulation of flowering in Brachypodium and wheat, and that this role is associated with the simultaneous regulation of other FT-like genes. The differential effects of mutations in FT-A1 and FT-B1 on wheat heading time suggest that different allelic combinations of FT1 homoeologs could be used to adjust wheat heading time to improve adaptation to changing environments. PMID:24718312
Lv, Bo; Nitcher, Rebecca; Han, Xiuli; Wang, Shuyun; Ni, Fei; Li, Kun; Pearce, Stephen; Wu, Jiajie; Dubcovsky, Jorge; Fu, Daolin
Thiazolidinediones inhibit alkaline phosphatase activity while increasing expression of uncoupling protein, deiodinase, and increasing mitochondrial mass in C3H10T1\\/2 cells
Although there are a number of cell lines committed to differentiate into brown adipocytes, the stem-cell origin of brown fat remains unclear. To address this problem, we explored the effects of various pharmacological agents on differentiation of C3H10T1\\/2 cells, a pluripotent stem-cell line of mesodermal origin. Histochemical and biochemical analysis revealed that, when these cells were treated with retinoic acid,
Mark A. Paulik; James M. Lenhard
Acute myocardial infarction: tissue characterization with T1rho-weighted MR imaging--initial experience.
Acute myocardial injury was evaluated in 21 patients by using a contrast material-enhanced T1rho-weighted cine turbo field-echo magnetic resonance (MR) imaging sequence and a delayed-enhancement sequence. In 12 of 21 patients, conventional T1-weighted contrast-enhanced cine turbo field-echo MR images were also collected for direct comparison with T1rho-weighted images. Delayed-enhancement technique distinctly characterized irreversible injury (percentage enhancement, 588% +/- 344). With T1rho weighting, percentage enhancement of irreversibly injured myocardium was 68% +/- 41, compared with 23% +/- 24 without T1rho weighting (P <.006). The addition of T1rho weighting to contrast-enhanced cine turbo field-echo MR sequences may offer a new contrast enhancement mechanism for characterization of acutely infarcted myocardium. PMID:15215547
Muthupillai, Raja; Flamm, Scott D; Wilson, James M; Pettigrew, Roderic I; Dixon, W Thomas
A Gal4/UAS system for conditional transgene expression in rhombomere 4 of the zebrafish hindbrain
Background The zebrafish is well established as a model organism for the study of vertebrate embryogenesis, but transgenic lines enabling restricted gene expression are still lacking for many tissues. Results We first generated the hoxb1a(?¢globin):eGFPum8 line that expresses eGFP in hindbrain rhombomere 4 (r4), as well as in facial motor neurons migrating caudally from r4. Second, we generated the hoxb1a(?-globin):Gal4VP16um60 line to express the exogenous Gal4VP16 transcription factor in r4. Lastly, we prepared the UAS(?-actin):hoxa3aum61 line where the hoxa3a gene, which is normally expressed in r5 and r6, is under control of Gal4-regulated UAS elements. Crossing the hoxb1a(?-globin):Gal4VP16um60 line to the UAS(?-actin):hoxa3aum61 line drives robust hoxa3aexpression in r4. We find that transgenic expression of hoxa3a in r4 does not affect hoxb1a expression, but has variable effects on migration of facial motorneurons and formation of Mauthner neurons. While cases of somatic transgene silencing have been reported in zebrafish, we have not observed such silencing to date ¢ possibly because of our efforts to minimize repetitive sequences in the transgenic constructs. Conclusion We have generated three transgenic lines that will be useful for future studies by permitting the labeling of r4-derived cells, as well as by enabling r4-specific expression of various transgenes.
Choe, Seong-Kyu; Nakamura, Mako; Ladam, Franck; Etheridge, Letitiah; Sagerstrom, Charles G.
Expression of human protamine P1 in sperm of transgenic mice
Transgenic mice were produced by pronuclear injection with DNA constructs containing human protamine P1 cDNA recombined with a murine protamine P1 promoter, and were identified by PCR. Expression of human P1 was investigated using huplm, a monoclonal antibody specific for human P1, applied to murine testicular cells, smears of epididymal sperm, and smears of detergent-isolated sperm nuclei. Various antibodies and nontransgenic littermates were used as controls. Two male founders (T3 and T7) sired more than five generations of transgenic offspring each with continued expression of human P1 in their sperm. Transgenic animals appear of normal fertility with sperm of typical nuclear morphology. The human P1 transgene was expressed postmeioticly in both lines, as expected. Nearly 100% of sperm of T3 and T7 hemizygotes labeled with huplm, consistent with complete diffusion of human P1 protein through the intercellular bridge of spermatogenic cells. Human P1 labeling of sperm nuclei was not visibly affected by sonication or by treatment with the detergent MATAB or the reducing agent DTT. A third founder female (T5) showed a transmission pattern consistent with insertion of the transgene into an X chromosome; her transgenic offspring expressed human P1 in only a small fraction of sperm. Human P1 transgenes may serve as efficient targets for germinal mutations and transgenicmice may provide promising models for investigating the DNA complexes.
Wyrobek, A.J.; Keith, C.; Stilwell, J.; Lowe, X. [Lawrence Livermore National Laboratory, CA (United States); Anderson, G. [Univ. of California, Davis, CA (United States)
Transgene integration and organization in cotton (Gossypium hirsutum L.) genome.
While genetically modified upland cotton (Gossypium hirsutum L.) varieties are ranked among the most successful genetically modified organisms (GMO), there is little knowledge on transgene integration in the cotton genome, partly because of the difficulty in obtaining large numbers of transgenic plants. In this study, we analyzed 139 independently derived T0 transgenic cotton plants transformed by Agrobacterium tumefaciens strain AGL1 carrying a binary plasmid pPZP-GFP. It was found by PCR that as many as 31% of the plants had integration of vector backbone sequences. Of the 110 plants with good genomic Southern blot results, 37% had integration of a single T-DNA, 24% had two T-DNA copies and 39% had three or more copies. Multiple copies of the T-DNA existed either as repeats in complex loci or unlinked loci. Our further analysis of two T1 populations showed that segregants with a single T-DNA and no vector sequence could be obtained from T0 plants having multiple T-DNA copies and vector sequence. Out of the 57 T-DNA/T-DNA junctions cloned from complex loci, 27 had canonical T-DNA tandem repeats, the rest (30) had deletions to T-DNAs or had inclusion of vector sequences. Overlapping micro-homology was present for most of the T-DNA/T-DNA junctions (38/57). Right border (RB) ends of the T-DNA were precise while most left border (LB) ends (64%) had truncations to internal border sequences. Sequencing of collinear vector integration outside LB in 33 plants gave evidence that collinear vector sequence was determined in agrobacterium culture. Among the 130 plants with characterized flanking sequences, 12% had the transgene integrated into coding sequences, 12% into repetitive sequences, 7% into rDNAs. Interestingly, 7% had the transgene integrated into chloroplast derived sequences. Nucleotide sequence comparison of target sites in cotton genome before and after T-DNA integration revealed overlapping microhomology between target sites and the T-DNA (8/8), deletions to cotton genome in most cases studied (7/8) and some also had filler sequences (3/8). This information on T-DNA integration in cotton will facilitate functional genomic studies and further crop improvement. PMID:17549600
Zhang, Jun; Cai, Lin; Cheng, Jiaqin; Mao, Huizhu; Fan, Xiaoping; Meng, Zhaohong; Chan, Ka Man; Zhang, Huijun; Qi, Jianfei; Ji, Lianghui; Hong, Yan
Comparison of T1c versus T2 prostate cancers in Japanese patients undergoing radical prostatectomy
In order to examine the characteristics of patients with nonpalpableprostate cancer (T1c cancer) in Japan, patients treated with radicalprostatectomy were compared with those with palpable (T2) cancer.Prostate-specific antigen (PSA) level in patients with T2b disease wassignificantly higher than those with T1c and T2a tumors. At the time ofradical prostatectomy, 78%, 71% and 31% of patients with T1c, T2a, and T2b,
Y. Furuya; S. Ohta; N. Sato; T. Kotake; M. Masai
Hyperplasia and tumours in lung, breast and other tissues in mice carrying a RAR beta 4-like transgene.
Transgenic mice were generated which express a truncated nuclear retinoic acid receptor beta (RAR beta), closely resembling the natural isoform RAR beta 4, under the control of the MMTV promoter. The transgene was expressed in salivary gland, testis, lung and mammary tissue in two different lines. At approximately 11-14 months virtually all the transgenic mice showed hyperplasia of the lung alveolar epithelium with an excess of type II pneumocytes. Hyperplasia of the mammary alveoli and terminal ducts was also seen in some females. Salivary glands and some sebaceous glands were hyperplastic in most male transgenic mice, but only rarely in females or in non-transgenics. Primary benign and malignant tumours were more numerous in transgenic mice than in controls, with a total of 23 in 43 mice versus two in 33 non-transgenic animals. Treatment with dexamethasone to increase transgene expression resulted in exaggerated versions of the above phenotypes. Overexpression of RAR beta 4 therefore appears to predispose various tissues to hyperplasia and neoplasia, and this by contrast to the RAR beta 2 isoform, which has tumour suppressor activity. A survey of ratios of RAR beta 4:RAR beta 2 expression in human lung tumour cell lines showed an increase compared with normal lung tissue, suggesting that RAR beta 4 may play a similar role in human tumorigenesis. Images
Berard, J; Gaboury, L; Landers, M; De Repentigny, Y; Houle, B; Kothary, R; Bradley, W E
Accelerated T1rho relaxation quantification in intervertebral disc using limited spin-lock times
Objective T1rho relaxation measurement has the potential to identify early biochemical changes in the intervertebral disc. Traditionally, multiple spin-lock times (SLT), often ~5 SLTs, are used to ensure the accuracy and robustness of T1rho mapping. It will be advantageous to use fewer SLT points if comparable accuracy of T1rho mapping can be achieved. In this study, the feasibility of using 3 SLT points to measure intervertebral disc T1rho relaxation time is explored. Materials and methods The lumbar spine of 12 subjects (age range: 30-75 years, disc =60) were studied on 3-T MRI. For T1rho measurement, a rotary echo spin-lock pulse was implemented in a 3D balanced fast field echo (b-FFE) sequence. Spin-lock frequency was set as 500 Hz and the SLTs of 1, 10, 20, 40, and 60 ms were acquired. T1rho maps were generated by fitting each pixelÆs intensity as a function of SLT using a non-negative least-square fitting algorithm. Images were analysed in the mid-sagittal section. T1rho maps were re-constructed using all 5 SLT points of 1, 10, 20, 40, and 60 ms, and three SLT points of 1, 20, and 60 ms respectively. ROIs included nucleus pulposus (NP), anterior annulus fibrosus (AF) and posterior annulus fibrosus. Values of anterior AF and posterior AF were averaged as the value for AF. Agreement of T1rho measurements using different SLT points was assessed using intra-class correlation coefficient (ICC) on absolute agreement as well as Bland and Altman plot. Results There was no significant difference for T1rho values by 5-SLT measurement and 3-SLT measurement in both NP (P=0.63) and AF (P=0.31). The ICC for 5-SLT T1rho measurement vs. 3-SLT T1rho measurement was 0.991 and 0.981 respectively for NP and AF T1rho time. The Bland and Altman plots for the comparison showed a mean difference of 3.14 and 1.83 for NP and AF respectively. Polling the T1rho values for NP and AF in 60 discs together, the ICC for 5-SLT T1rho measurement vs. 3-SLT T1rho measurement was 0.993, and the Bland and Altman analysis showed a mean difference of 2.56. Conclusions This study suggests that adopting 3 SLTs of 1, 20, and 60 ms can be an acceptable alternative for the disc T1rho measurement.
Zhao, Feng; Yuan, Jing; Mok, Greta SP; Ahuja, Anil T; Griffith, James F
Measurement of T1 of human arterial and venous blood at 7T
Techniques for measuring cerebral perfusion require accurate longitudinal relaxation (T1) of blood, a MRI parameter that is field dependent. T1 of arterial and venous human blood was measured at 7T using three different sources ¢ pathology laboratory, blood bank and in vivo. The T1 of venous blood was measured from sealed samples from a pathology lab and in vivo. Samples from a blood bank were oxygenated and mixed to obtain different physiological concentrations of hematocrit and oxygenation. T1 relaxation times were estimated using a three-point fit to a simple inversion recovery equation. At 37░ C, the T1 of blood at arterial pO2was 2.29 ▒ 0.1 s and 2.07 ▒ 0.12 at venous pO2. The in vivo T1 of venous blood, in three subjects, was slightly longer at 2.45 ▒ 0.11s. T1 of arterial and venous blood at 7T was measured and found to be significantly different. The T