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Nets 104, Cavs 99
--I'm not going to spend a lot of time tonight because the wireless at the Izod Center is a huge joke. Like most things with the Nets and the Meadowlands complex, it is sub-standard. --The Cavs lost this game in the first quarter. They had a chance to survive it, but they lost that at the start of the third quarter when they gave up a 10-2 run. All the stuff that happened in the fourth -- the comeback that fell short -- is window dressing. The Cavs went to their small lineup and played junk ball and it worked in the short term. At the end, everybody got tired and there were silly mistakes that finished them. But that's what happens when you get down 19, you have to be perfect to come back. Sometimes it's not too late, tonight it was. When they finish with 46 or 47 wins this will be one of the games where they lost a chance at 50. --This is the 15th different starting lineup the Cavs have used this season, there are going to be consequences when that happens. Joe Smith and Delonte West have never played with Sasha Pavlovic. It showed, they covered the pick-and-roll and and rotated like they were running in sand. Anderson Varejao was simply terrible on defense in the first half and LeBron got caught on rotations. After the first quarter, the Cavs held the Nets to 41 percent shooting. But the first quarter counts, too. --I personally question whether Sasha is really ready. He's only had a couple practices, it's a very serious injury and he missed more than a quarter of the season. That is a lot to overcome, I think you may need more than two days of contact. --Let me say, the Nets played great, especially with their ball movement early. But down the stretch, they reverted to form. They are not a good team, though you saw Devin Harris' value late. He was 1-of-22 on 3-pointers on their five-game road trip so you knew he was probably due to make a couple. --Devin Brown and Damon Jones played excellent tonight. Devin shut down Vince Carter and Damon really held his own at the defensive end. He got beat a couple of times by Devin Brown, but you have to expect that. These two guys work hard, really are committed to running the offense, and are veterans. No way they should lose playing time, though they shouldn't play this much. Also, Wally Szczerbiak played well tonight. He missed some open jumpers, but he's been doing that. He had five assists and was good on defense when he was in there. Based on the way Sasha, Delonte and Joe Smith played, Wally was good tonight. --LeBron's attack to this game was top notch, he was relentless in going to the basket and it got him 21 free throws. He made just one 3-pointer and had 42 points, usually he's got to be hot from the outside to go for 40. --The Cavs and especially LeBron, who played a whopping 46 minutes tonight, expended a lot of energy in this loss. It'll probably show up tomorrow. --After the game in the locker room, Austin Carr when up and congratulated LeBron on passing him for second place on the Cavs scoring list. By next week, LeBron will probably pass Brad Daugherty to become the all-time leader. LeBron was very gracious and pleased that A.C. would come and personally give him the best wishes. LeBron's sense of history is remarkable, he really is a student of the game and, unlike some young players, he goes out of his way to pay respect to the elders. That's not a stroke job either, I've seen him do it many times and it is sincere on his behalf.
Starting linueps
Cavs: Delonte West, Sasha Pavlovic, LeBron James, Joe Smith, Anderson Varejao Nets: Devin Harris, Vince Carter, Richard Jefferson, Nenad Kristic, Josh Boone
Officials Dick Bavetta, Violet Palmer, Gary Zielinski.....I think this is just the first or second time the Cavs have had Bavetta this season. He likes to call charges.
--Yes, that is Sasha Pavlovic in the starting lineup. While this may seem crazy, taking a guy who hasn't played in 23 games and sticking him right in the fire, this actually sticks to Mike Brown form. With the exception of when LeBron came off the bench to protect Varejao from getting booed in December, Mike wants to know right away whether a guy is on his game or not. Which is why we've seen guys go from the inactive list to the starting lineup in the past. Of course, don't tell that to Devin Brown, who had been playing great and is now coming off the bench. --Second thing with the starters is that Ben Wallace is out with back pain. The injury report says he has back spasms, I think Mike slipped before the game and said he had a back strain. Maybe this is the same thing, but I think there is a difference. Anyway, the team sounded optimistic Ben would play yesterday and now they sound pessimistic about tomorrow in D.C. For what it is worth. Joe Smith is in there, which is the dynamic I think the Cavs need. A scorer and a defender in the post. Be it a Wallace/Zydrunas Ilgauskas pairing or Smith/Varejao or whatever. Either way, Dwayne Jones is going to have to play some minutes tonight and LeBron will have to play some at power forward I suspect. --The Nets are just back from a West Coast trip in which they went 0-5. They have been really struggling to score since trading Jason Kidd and Harris can't seem to buy a shot. But the Cavs have not played well here in the regular season over the last couple years. They are 0-2 against the Nets this season.
Halftime -- Nets 58, Cavs 47 --The Cavs were very lethargic at the outset of this game, a rather cocky attitude that they thought they were going to just show up an win. The Nets did make 14 of their 16 shots and they made a lot of jumpers, too. But most of those baskets came around the basket as Anderson Varejao especially was terrible. The Nets have 30 points in the paint at half, which is the real reason why they are ahead. --There was a bit of a comeback in the second quarter when the Cavs were more active and not some defensive stops. But they again got a little loose late with the defense. The Nets are not a good offensive team and they will miss lots of jumpers if you give them the chance. But they have 19 assists in the half because they are just throwing it in the paint and getting hoops. --That said, the Cavs offensive has been pretty strong. They had assists on their first 13 baskets and there's lots of driving to get fouls, especially from LeBron, who is being very aggressive on that end of the floor. If that continues they will have a chance to win this game, but it will be predicated on whether they can get stops. --Sasha played six very average minutes before being pulled, missing two wide open jumpers. Maybe as a result both Devin Brown and Wally Szczerbiak played well of the bench. Sasha's leash will probably shorter in the second half.


text:
|Publication number
|Publication type
|Application number
|Publication date
|Feb 11, 1997
|Filing date
|May 8, 1995
|Priority date
|Oct 4, 1993
|Fee status
|Publication number
|Kathryn M. Albers, Brian M. Davis
|Original Assignee
|University Of Kentucky Research Foundation
|Export Citation
|BiBTeX, EndNote, RefMan
|Patent Citations (9), Non-Patent Citations (58), Referenced by (7), Classifications (18), Legal Events (4)
|External Links: USPTO, USPTO Assignment, Espacenet
This application is a continuation of application Ser. No. 08/131,424 filed Oct. 4, 1993, now abandoned.
The present invention relates to transgenic mice that express increased levels of nerve growth factor (NGF) in the epidermis and other stratified, keratinized epithelium. The nerve growth factor expressing transgenic mice of the present invention are valuable to researchers that study the role of nerve growth factor and other neurotrophic factors in the development and function of the nervous system, and in the study of neurodegenerative disorders of the brain such as Parkinson's syndrome and Alzheimer's disease.
The survival of developing vertebrate neurons is thought to be dependent on access to a limited supply of target-derived neurotrophic factors. Nerve growth factor (NGF) is the most extensively characterized of these factors and has been shown to regulate the survival of discrete populations of neurons.
U.S. Pat. No. 4,185,095 to Young discloses the isolation of a nerve growth factor from mouse submandibular or salivary gland.
U.S. Pat. No. 5,082,774 to Heinrich discloses a recombinant human nerve growth factor. The nerve growth factor is in an expression vector with an adenovirus strong, major, late promoter. Heinrich discloses that the expression vector may be used to stably transfect mammalian cells such as CHO or COS cells.
U.S. Pat. No. 5,180,820 to Barde et al. discloses that NGF has been molecularly cloned.
Various transgenic mice have been patented. U.S. Pat. No. 4,736,866 to Leder et al. discloses a transgenic non-human eukaryotic animal whose germ cells and somatic cells contain an activated oncogene sequence introduced into the animal or an ancestor of the animal at an embryonic stage. The embryo of the mice were microinjected with approximately 500 copies of the RSVS-107 c-myc plasmid. The injected eggs were transferred to pseudopregnant foster families and allowed to develop to term. The plasmid contains a Rous Sarcoma Virus enhancer and promoter sequence. The resultant mice showed expression of the c-myc gene in the salivary gland, spleen, testes, lung, brain, and preputial gland and intestinal tissue.
U.S. Pat. No. 5,175,383 to Leder et al. discloses a male transgenic mouse containing germ cells and somatic cells which contain a recombinant gene which is a vertebrate gene in the Int-2/FGF family which is capable of promoting benign prostatic hyperplasia or hypertrophy. The fusion gene which is injected into the mouse embryonic tissue comprises a promoter sequence controlling transcription of the recombinant gene such as a promoter derived from the mouse mammary tumor virus and cytomegalovirus. The recombinant gene is preferably substantially homologous with (i.e., greater than 50% homologous in terms of encoded amino acid sequence) a naturally occurring, vertebrate gene in the Int-2/FGF gene family of murine growth factor encoding genes or their vertebrate counterparts, including the murine acidic or basic fibroblast growth factor genes, the murine FGF-5 gene, the murine epidermal growth factor gene, the murine insulin-like growth factor-1 and -2 gene, the murine α-transforming growth factor gene with a murine hst/KS3 gene. The transgenic mice of Leder et al. exhibit prostate hyperplasia and give sterile offspring.
U.S. Pat. No. 5,175,384 to Krimpenfort et al. discloses transgenic mice having a phenotype characterized by the substantial absence of mature T-lymphocytes. The mouse is produced by introducing a transgene into a zygote of a mouse which comprises gene fragment which encodes a T-cell antigen receptor polypeptide variant which is incapable of mediating T-cell maturation in the transgenic mouse.
U.S. Pat. No. 5,175,385 to Wagner et al. discloses the production of a transgenic mouse with enhanced viral resistance which is transmissible to its offspring. The transgenic mouse is prepared by introduction of a gene encoding a human interferon having anti-viral activity into a host mouse. The plasmid of Wagner et al. contains a methallothionein-1 promoter and the genomic human beta interferon gene. When these mice were injected with pseudorabies virus they showed an increased resistance to the virus and although many mice died they died considerably later than did the control animals.
Vassar et al., in the Proceedings of the National Academy of Sciences, Volume 86, pages 1563-1567, March 1989, disclose "Tissue-specific and Differentiation-specific Expression of a Human K14 Keratin Gene in Transgenic Mice". This publication discloses that Vassar et al. used a plasmid containing a K14 keratin promoter and a neuropeptide substance P and determined the expression of the plasmid and neuropeptide substance P in the tail skin of transgenic mice.
Vassar et al. in Gene & Development, Volume 5, pages 714-727, (1991) disclose "Transgenic Mice Provide New Insights into the Role of TGF-α During Epidermal Development and Differentiation". This publication discloses the construction of transgenic mice using a plasmid which contains the K14 keratin promoter and the TGF-α gene. Vassar et al. disclose that the TGF-α gene belongs to the epidermal growth factor family of proteins and shows structural homology with the epidermal growth factor. These transgenic mice showed gross phenotypic abnormalities in their skin including flaky outer epidermal layers with stunted hair growth and wrinkling. The plasmid used to create this transgenic mouse also included human growth hormone fusion gene.
Patil et al. in Neuron, Volume 2, pages 347-447, March 1990, disclose "Specific Neuronal Expression of Human Nerve Growth Factor Receptors in the Basal Forebrain and Cerebellum of Transgenic Mice". Patil et al. employed a cosmid clone containing the entire NGF receptor gene. Introduction of the human NGF receptor gene into the mouse germ line resulted in significant levels of expression in specific cells in both the peripheral and central nervous systems. The NGF receptor gene and the NGF gene are distinct genetic material.
Borrelli et al. in Proceedings of the National Academy of Science, Volume 89, pages 2764-2768, April 1992, disclose "Pituitary Hyperplasia Induced by Ectopic Expression of Nerve Growth Factor". Borrelli et al. fused nerve growth factor cDNA to rat prolactin promoter to induce its ectopic expression in pituitary lactotrophs of transgenic mice.
Federoff et al. in Proceedings of the National Academy of Science, USA, Volume 89, pages 1636-1640, March 1992, disclose "Expression of Nerve Growth Factor in Vivo from a Defective Herpes Simplex Virus-1 Vector Prevents Effects of Axotomy on Sympathetic Ganglia". This publication teaches that rat nerve growth factor packaged into a herpes simplex virus-1 particle after axotomy of sympathetic superior cervical ganglion prevented a decline in the tyrosine hydroxylase level which is generally required for maintenance of the noradrenergic neurotransmitter system.
Edwards et al. in the Cell, Volume 58, pages 151-170, Jul. 14, 1989, disclose "Directed Expression of NGF to Pancreatic β Cells in Transgenic Mice Leads to Selective Hyperinnervation of the Islets". Edwards et al. showed a selective increase the expression of NGF in pancreatic β cells by creating a hybrid gene that contained a rat insulin promoter fused to mouse NGF cDNA followed by an intron and transcriptional terminator from the SV40 virus.
PCT Publication WO 93/00909 is directed to a method of treating neurotrophin-expressing tumors by administering a sequence complementary to at least a portion of a RNA transcript of brain-derived neurotrophic factor gene.
During development of the vertebrate nervous system up to half of all neurons generated undergo a process of naturally occurring cell death (See Ard, M. D., Morest, D. K. (1984). Cell death during development of the cochlear and vestibular ganglia of the chick. Intl. J. Dev. Neurosci. 2, 535-547; and Oppenheim, R. W. (1991). Cell death during development of the nervous system. Ann. Rev. Neurosci. 14, 453-501).
Neuronal death typically occurs shortly after neurons being making functional connections within their target field. Survival during this period of innervation is thought to be dependent on the synthesis by the target tissue of a limited quantity of neurotropic substances. (See Thoenen, H. and Barde, Y. A. (1980). Physiology of nerve growth factor. Physiol. Rev. 60, 1284-1334; Oppenheim, R. W. (1989). The neurotrophic theory and naturally occurring motoneuron death. Trends in Neurosci. 12, 252-255; and Davies, A. M., Larmet, Y., Wright, E., Vogel, K. S. (1991). Coordination of trophic interactions by separate developmental programs in sensory neurons and their target fields. J. Cell Sci. 15, 111-116)).
The competition for- target field-derived neurotrophic factors is though to serve at least two functions: to ensure that an appropriate number of synaptic contacts are made at the target and to eliminate inappropriate neuronal projections. (See Oppenheim, R. W. (1981). Neuronal death and some related phenomena during neurogenesis. In Studies in Developmental Biology (ed. W. M. Cowan) p 74-133. Oxford University Press; and Cowan, W. M., Fawcett, J. W., O'Leary, D.D.M., and Stanfield, B. B. (1984). Regressive events in neurogenesis. Science 225, 1258-1265)).
This concept is referred to as the neurotrophic hypothesis and predicts that the number of neurons in the adult could be increased if a higher concentration of neurotrophic factor(s) was available during the critical time when synaptic contacts are being established.
Nerve growth factor (NGF) is a prototypical target-derived neurotrophic substance that has been shown to be essential for the survival and differentiation of neural crest-derived sensory neurons, sympathetic neurons, and forebrain cholinergic neurons. (See Levi-Montalcini, R. and Booker B. (1960). Destruction of the sympathetic ganglia in mammals by an antiserum to a nerve-growth protein. Proc. Natl. Acad. Sci. U.S.A 46, 384-391 and Hefti, F. (1986) J. Neurosci. 6, 2155-2161; Williams, L. R., Varon, S., Peterson, G., Wictorin, K., Fischer, W., Bjorklund, A. & Gage, F. H. (1986). Proc. Natl. Aced. Sci. U.S.A. 83, 9231-9235).
The role of NGF in neuron survival is supported by numerous studies that have utilized both tissue culture and in vivo approaches in which NGF peptide or NGF antibodies were applied. (See Levi-Montalcini and Booker, 1960, supra; Johnson, E. M., Gorin, P. D., Brandeis, L. D., Pearson, J. (1980). Dorsal root ganglion neurons are destroyed by exposure in utero to maternal antibody to nerve growth factor. Science 210, 916-918; Barde, Y. A., Edgar, D., Thoenen H. (1980). Sensory neurons in culture: Changing requirements for survival factors during embryonic development. Proc. Natl. Acad. Sci. U.S.A. 77, (1980) 1199-1203; Hamburger, V. and Yip, H. W. (1984). Reduction of experimentally induced neuronal death in spinal ganglia of the chick embryo by nerve growth factor. J. Neurosci. 4, 767-774). Sympathetic and certain sensory neurons can be reduced by exogenous NGF whereas anti-NGF antibodies eliminate these neurons.
During development of skin, NGF is expressed by target cells of the presumptive epidermis and dermis. (See Davies, A. M., Bandtlow, D., Heumann, R., Korsching, S., Rohrer, H., and Thoenen, H. (1987). Timing and site of nerve growth factor synthesis in developing skin in relation to innervation and expression of the receptor. Nature 326, 353-358).
Target-field synthesis of NGF coincides with the arrival of axons to the epidermis, though the onset of expression as well as the concentration of NGF are independent of innervation. (See Rohrer, H., Heumann, R., Thoenen, H. (1988). Synthesis of nerve growth factor (NGF) in developing skin is independent of innervation. Dev. Biol. 128, 240-244; and Harper, S. J., and Davies, A. M. (1990). NGF mRNA expression in developing cutaneous epithelium related to innervation density. Development 110,515-519). Thus, cells of the target field appear to control the onset and level of NGF gene expression.
In mouse whisker pad skin, time course studies of innervation have shown that by embryonic day 11 (E11) axons have grown out to the skin and NGF mRNA expression has begun. (See Davies et al., 1987, supra). NGF continues to rise until E14 and then falls sharply by E15. The decrease in NGF concentration coincides with the onset of death of nearly half of the neurons in the trigeminal ganglion.
As skin innervation proceeds, the embryonic epithelium begins the transformation from a two-cell layer, undifferentiated periderm to a multilayer, stratified epidermis. (See Schweizer, J., and Winter, H. (1982). Keratin polypeptide analysis in fetal and in terminally differentiating newborn mouse epidermis. Differentiation 22, 19-24; and Kopan, R. and Fuchs, E. (1989). A new look into an old problem: Keratins as tools to investigate determination, morphogenesis, and differentiation in skin. Genes and Devel. 3, 1-15).
The differentiation of the epidermis is marked by the expression of epidermal specific genes, one of the earliest being the gene encoding the keratin intermediate filament protein K14. The onset of K14 gene expression in mouse skin occurs at approximately E14-E15 (see FIG. 1(b), beginning as NGF concentration is declining.
Heretofore there has been no convenient method to investigate how target-derived NGF affects neuron survival and development of the peripheral nervous system or provide a practical model for testing drug candidates for the treatment of neurodegenerative disorders of the brain such as Parkinson's syndrome and Alzheimer's disease.
The present invention overcomes the deficiency by providing transgenic mice that manufacture increased levels of NGF in the basal keratinocytes of the skin and exhibit hyperinnervation.
The invention provides a transgenic mouse whose somatic and germ cells contain and express a gene encoding for nerve growth factor. The gene exhibits hyperinnervation in the mouse, and has been introduced into a mouse or an ancestor of the mouse at an embryonic stage. The NGF gene is operatively linked to a human K14 keratin promoter and enhancer region.
The invention also provides for a mouse wherein the NGF gene has been introduced into an ancestor if said mouse at an embryonic stage by microinjection.
In an additional embodiment, the mouse nerve growth factor is further operatively linked to the human growth hormone gene.
The invention provides for a mouse which has a phenotype characterized by hyperinnervation of the skin, hypertrophy of trigeminal ganglion and enlargement of the superior cervical sympathetic and dorsal root ganglia.
Advantageously, the present invention provides a method of producing the transgenic mouse which overexpresses nerve growth factor which comprises
(a) providing a mouse nerve growth factor gene operatively linked to a human K14 keratin promoter and enhancer region functional in said mouse;
(b) introducing said gene into a mouse embryo;
(c) transplanting said embryo into a pseudopregnant mouse; and
(d) allowing said embryo to develop to term.
Furthermore, the invention provides a method of testing the efficacy of drugs in treating neurodegenerative disorders comprising administering said drug to a mouse according to the invention, and determining the behavioral effects and tissue changes of said drug on said mouse.
FIGS. 1a, b, c: (FIG. 1(a) shows the K14-NGF fusion gene contains the mouse NGF cDNA cloned downstream from the human epidermal K14 gene promoter and enhancer sequences.
FIG. 1(b) shows a schematic depicting the onset of NGF expression by E11, and its rise and fall at E15.
FIG. 1(c) shows mice screened for the transgene by Southern hybridization of DNA isolated from tail samples obtained three to four weeks after birth. Blots were probed using a 32 P-labeled DNA sequence complementary to the mouse NGF cDNA.
FIGS. 2a, b: FIG. 2(a) examines the expression level of the K14-NGF transgene for transgenic mouse lines. Shown is a 3 d exposure, FIG. 2(a) and a 10 d exposure, FIG. 2(b), of the same filter. FIG. 2(b) examines the tissue specificity of K14-NGF expression.
FIGS. 3a-d: FIG. 3 shows cellular expression of the K14-NGF transgene in skin from A-56 F1 transgenics was examined by in situ hybridization. Basal cells (arrows) in whisker pad epidermis, FIG. 3(a), and vibrissa, FIG. 3(c), of control animals had silver grain density at background levels. A-56 F1 transgenic mice had a greater labeling density of whisker pad basal cells, FIG. 3(b) (arrows), and vibrissa, FIG. 3(d).
FIG. 4 shows expression of K14-NGF causes hyperinnervation of skin. Skin from dorsum, (FIGS. 4a and 4b) and whisker pad (FIG. 4c and 4d) was processed for paraffin histology and sections stained with hematoxylin and eosin. Control skin (FIG. 4a and 4c) show few apparent neuron bundles whereas A-56 F1 transgenic skin (FIG. 4b and 4d) shows numerous, enlarged fiber bundles.
FIGS. 5a-d: FIG. 5 shows immunolabeling of transgenic skin reveals a hyperinnervation of sensory and sympathetic neurons. To verify that the fiber bundles in the skin were composed of neuronal processes, frozen sections of whisker pad skin from control (FIG. 5a) and A-56 F1 transgenics (FIG. 5b) were immunolabeled with an anti-neurofilament 150 antibody followed by incubation with a goat anti-rabbit FITC conjugated secondary. A significant increase in the number of fibers and bundles was seen in an equivalent region of A-56 transgenic skin (FIG. 5b). A-56 skin contained sympathetic fibers, sections were immunolabeled with an anti-TH antibody followed by a biotinylated goat-anti rabbit secondary processing with DAB (FIG. 5c). A-56 F1 transgenics labeled with anti-CGRP followed by a biotinylated goat-anti rabbit secondary antibody are shown in FIG. 5d.
FIGS. 6a, b: FIG. 6 shows expression of K14-NGF causes enlargement of the trigeminal and superior cervical ganglia. Trigeminal ganglia (FIG. 6a) and superior cervical ganglia (FIG. 6b) were removed from control A-48 F1, and A-56 F1 mice.
The plasmid containing k14-NGF-hGH has been deposited with the American Type Culture Collection under the terms of the Budapest Treaty and is available as Accession No. 69423.
The present invention provides transgenic mice that manufacture increased levels of nerve growth factor (NGF) in the basal keratinocytes of the skin.
The mice are obtained by making a fusion gene construct in which the human K14 keratin promoter and enhancer region was linked to mouse NGF cDNA.
To increase the expression of the transgene, a region from the human growth hormone gene may, in a preferred embodiment, be added to the construct. Production of nerve growth factor in the epidermis of these mice was found to result in a profound hyperinnervation of the peripheral nervous system, i.e. skin has increased nerve fibers, the trigeminal sensory, the superior cervical sympathetic, and the dorsal root ganglia were markedly enlarged.
The nerve growth factor expressing transgenic mice of the present invention are valuable to researchers that study the role of nerve growth factor and other neurotrophic factors in the development and function of the nervous system, and in the study of neurodegenerative disorders of the brain such as Parkinson's syndrome and Alzheimer's disease.
To investigate the role of NGF in the development of the peripheral nervous system and skin innervation, the human K14 gene promoter was used to drive the expression of the NGF cDNA in basal keratinocytes. Results show that target-derived overexpression of NGF has profound effects on the level of skin innervation and neuronal survival.
Below is a more detailed outline of experimental procedure. NGF cDNA was linked to the promoter and enhancer regulatory regions of the human keratin 14 gene. The K14-NGF fusion gene was expressed at approximately embryonic day 14, the time at which endogenous NGF levels begin to decline and programmed neuronal death begins. In situ hybridization of skin of K14-NGF transgenics had increased levels of NGF mRNA in the basal cells of the epidermis and associated hair follicle structures. The increase in target derived NGF mRNA was correlated with a hypertrophy of peripheral sensory and sympathetic nerves, hyperinnervation of the skin, and a 26-117% increase in the number of primary sensory neurons in the trigeminal ganglion.
These results demonstrate that the level of NGF production by the target tissue dictates the level of neuron survival, and in so doing, influences the distribution and number of neurons in the adult.
NGF was overexpressed in the epidermis of transgenic mice using a fusion gene construct in which the human epidermal K14 keratin promoter and enhancer sequences were linked to a mouse NGF cDNA (FIG. 1a). The K14 promoter has previously been shown to direct high level of expression of various transgenes to basal keratinocytes of the epidermis. (See Vassar, R., Rosenberg, M., Ross, S., Tyner, A. & Fuchs, E. (1989). Tissue-specific and differentiation-specific expression of a human K14 keratin gene in transgenic mice. Proc. Natl. Acad. Sci. 86, 1563-1567; and Vassar, R., Coulombe, P. A., Degenstein, L., Albers, K. & E. Fuchs (1991). Mutant keratin expression in transgenic mice causes marked abnormalities resembling a human genetic skin disease. Cell 64, 365-380).
The rationale in designing the K14-NGF construct is based on the temporal overlap of NGF down-regulation with the initiation of K14 expression, i.e., as the concentration of target derived NGF beings to fall and neuronal cell death commences (E14-E15, see FIG. 1(b)), the K14-NGF transgene turns on, producing elevated levels of NGF during the critical period of cell death and thereafter in the adult (FIG. 1b).
Three founder mice have been isolated that express the transgene (FIG. 1c). Founder A-39 (FIG. 1c, lane 1) and founder A-56 (lane 3) were smaller and exhibited a lower rate of weight gain when compared to their nontransgenic siblings, whereas founder A-48 was normal in size. F1 offspring of founder A-56 also exhibited reduced weight gain. Founder A-39 has not generated transgenic offspring, though approximately eighty offspring have been screened. The A-56 transgenics also had delayed eye opening (at least 2-3 days later than nontransgenic siblings) and frequently exhibited a "squinty" appearance. This phenotype does not appear to be due to an increase in light sensitivity since even under low level lighting they retained a squinting appearance.
The relative level of K14-NGF expression in the three founders was determined using Northern hybridization analysis of total RNA isolated from full thickness skin (FIG. 2). Nontransgenic control skin samples (FIG. 2, lane 1) had a single faint band hybridizing with the radiolabeled NGF probe at approximately 1.3 kb, a size corresponding to the endogenous RNA transcript.
RNA samples isolated from the A-39 (lane 2), A-48 (lane 3), and A-56 (lane 4) skin contained, in addition to the endogenous transcript, a 1.9-kb band that corresponded to the predicted size of the K14-NGF transcript. Line A-39 and A-48 had comparable levels of transgene expression whereas expression in the A-56 line was significantly higher. Previous studies using the K14 promoter and enhancer region to express other transgenes of interest have shown transgene expression restricted to stratified, keratinized tissues such as skin, tongue, esophagus, and forestomach.
To verify that the tissue distribution of K14-NGF expression was comparable, RNA was analyzed from tongue, brain, heart, liver, and kidney of an A-56 F1 transgenic and nontransgenic control. Expression of the K14-NGF transgene was restricted to the keratinized epithelium samples examined, skin (FIG. 2, lane 5) and tongue (lane 6). The endogenous NGF transcript was detectable in skin (lanes 5 and 11), tongue (lanes 6 and 12), brain (lanes 7 and 13), heart (lanes 8 and 14), and kidney (lanes 10 and 16) of both the control and transgenic tissues. Liver tissues (lanes 9 and 15) showed no evidence of NGF expression.
Cellular expression of the K14-NGF transgene was examined using in situ hybridization on sections of whisker pad skin, a region known to have a high level of innervation (FIG. 3). A 35 S-labeled cRNA probe specific for the mouse NGF mRNA was hybridized to skin sections of 4 week-old K14-NGF transgenics and non-transgenic siblings and processed for autoradiography. Few autoradiographic grains were observed in the control skin (FIG. 3a) whereas the K14-NGF skin showed dense hybridization in the epidermal basal cell layer with decreasing intensity in suprabasal layers (FIG. 3b). Dense hybridization was also found in epidermal cells of the outer root sheath of hair and vibrissa follicles of transgenic mice (FIG. 3d), but not in the corresponding areas of nontransgenic (FIG. 3c). The epidermal and hair follicle distribution of hybridization is consistent with the known expression pattern of the K14 keratin (Schweizer and Winter, 1982; Kopan and Fuchs, 1989).
To determine whether the increased level of NGF mRNA in the transgenic epidermis was processed into functional NGF peptide, the skin of the transgenics was examined for evidence of increased neuronal survival. Histological examination of skin from various regions of the body (dorsum, ventral, tail, ear locations) revealed an increase in nerve processes and an abundance of large nerve bundles coursing throughout the papillary and reticular layers of the dermis (FIG. 4a, control; 4b, transgenic).
To confirm that these structures were composed of neuronal processes, whisker pad skin from A-56 F1 mice was immunolabeled with an antibody directed against the 150 Kd neurofilament protein (FIG. 4c, control; 4d, transgenic). In control mice, neurofilament positive fibers and bundles were evident in the reticular and deep layers of the dermis. Occasional small positive fibers could also be found penetrating the lower layers of the epidermis and in association with hair follicle and vibrissa structures.
A striking increase in the density and size of neuronal processes was apparent in skin of A-56 transgenics (FIG. 4d). Hyperinnervation was clearly evident in the papillary and reticular layers of the dermis as well as around hair follicle structures. Hyperinnervation was also apparent in skin of A-48 F1 mice, though at a reduced level than in the A-56 transgenics.
Two primary sources of axons for the observed whisker pad hyperinnervation are sensory neurons originating in the trigeminal ganglion and sympathetic postganglionic fibers arising from the superior cervical ganglion. As a first step in determining the contribution from these two sources, immunocytochemistry was performed using antibodies directed against the neurotransmitter calcitonin gene related peptide (CGRP), for identification of sensory neurons and the catecholamine-synthesizing enzyme tyrosine hydroxylase (TH), for identification of postganglionic sympathetic axons. (See Gibbins, I. L., Furness, J. B., Costa, M., MacIntyre, I., Hillyard, C. J. and Girgis, S. (1985). Colocalization of calcitonin gene-related peptide-like immunoreactivity with substance P in cutaneous, vascular and visceral sensory neurons of guinea pigs. Neurosci. Lett. 57, 125-130).
CGRP is enriched in sensory nerves projecting to hair follicles and as free endings in the epidermis. (See Kruger, L. Silverman J. D., Mantyh P. W., Sternini, C., Brecha, N. C. (1989). Peripheral patterns of calcitonin gene-related peptide general somatic sensory innervation: cutaneous and deep terminations. J. Comp. Neurol. 280, 291-302; and Shotzinger, R. J., and Landis, S. C. (1990). Postnatal development of autonomic and sensory innervation of thoracic hairy skin in the rat. Cell Tissue Res. 260, 575-587).
Though CGRP staining has been reported in sympathetic ganglion, it is thought to represent afferent input, not CGRP production by ganglionic neurons. In control skin (FIG. 5a), few CGRP-positive fibers could be identified. In contrast, the K14-NGF transgenic skin had numerous small and large bundles of CGRP-labeled fibers throughout the dermis, particularly in regions beneath the epidermis where abundant axons terminations occur (FIG. 5b).
Sympathetic innervation of transgenic skin was enhanced as shown by an increase in TH-immunoreactive fibers (FIG. 6c, control; 6d, transgenic). Much of the increased TH labeling was on fiber bundles in the upper regions of the dermis that also labeled for CGRP. TH-positive fibers appeared different from CGRP positive fibers however, in being thin, with numerous swellings. CGRP fibers were thicker and more uniform. Further distinction between CGRP labeled and TH labeled fibers was found in examination of Merkel cells of vibrissa structures. (See Nurse, C. A., Mearow, K. M., Homes, M., Visheau, B., and Diamond, J. (1983). Merkel cell distribution in the epidermis as determined by quinacrine fluorescence. Cell Tissue Res. 228, 51-54). Sensory, but not sympathetic neurons, recognize and associate with Merkel cells. (See Vos, P., Stark, F., and Pittman, R. N. (1991). Merkel cells in vitro: Production of nerve growth factor and selective interactions with sensory neurons. Dev. Biol. 144, 281-300). In transgenic and control samples only CGRP labeled fibers could be found associated with Merkel cells of the whisker follicle whereas no TH labeling was evident.
It was found that TH and CGRP synthesis is enhanced by NGF. No difference in TH-labeling could be discerned between the control and transgenic samples in the deep dermis. Few TH-immunoreactive fibers could be found in the deep dermis and these fibers were frequently associated with blood vessels that were adjacent to the underlying muscle layer, a location consistent with their sympathetic nature.
It was also found that NGF induces hypertrophy of trigeminal ganglion. The availability of elevated NGF peptide to neurons in the epidermal target field of the transgenics was further indicated by a striking hypertrophy of the trigeminal ganglion and the branches emanating from it. Cell counts made of ganglia isolated from three A-56 F1 transgenics had values that ranged from 26 to 117% greater than nontransgenic age-matched controls (Table 1 below). The hypertrophy of the trigeminal ganglion was also reflected by the enlargement of the primary branches of the trigeminal nerves. In the maxillary division of the trigeminal nerve dissected from control and transgenic samples, the transgenic nerve bundle (branch) is clearly larger than the control.
TABLE 1______________________________________ Number of PercentCase Number Age Neurons Difference______________________________________0401 Non-transgenic 24 d 26,054 550401 K14-NGF transgenic 24 d 40,2850527 Non-transgenic 29 d 26,493 1170527 K14-NGF transgenic 29 d 57,7670513 Non-transgenic 36 d 28,867 260513 K14-NGF transgenic 36 d 36,562______________________________________
A transgenic mouse model system was used to study the role of target field-derived NGF in development of the peripheral nervous system. Elevation of the production of NGF by the epidermal target tissue caused a dramatic increase in neuronal survival as evidenced by a striking hyperinnervation of the skin and hypertrophy of the trigeminal ganglia. Both sensory and sympathetic neurons in the transgenic skin appeared to be rescued by the increased NGF production as evidenced by enhanced CGRP- and TH-immunoreactivity. That both sensory and sympathetic neuron populations had increased survival is consistent with the known dependence of these cells on NGF. Sensory neurons are most dependent during embryonic development (shortly before birth) whereas sympathetic neurons require NGF within a 304 week period after birth. (See Levi-Montalcini and Booker, 1960, supra; Johnson et al., 1980, supra).
Since expression of the K14-NGF transgene begins at E14-E15, rises, and continues at a steady level in the adult, both sensory and sympathetic neurons would be exposed to elevated NGF at critical times in their development. Nerve terminals of both sympathetic and sensory neurons would presumably have access to a greater amount of NGF produced by the transgenic epidermis and transport it from the skin to cell bodies. Both sensory and sympathetic nerves have been shown to take up labeled NGF and transport it retrogradely. (See Hamburger, V., Brunso-Bechtold, J. K., Yip, J. W. (1981). Neuronal death in the spinal ganglia of the chick embryo and its reduction by nerve growth factor. J. Neurosci. 1, 60-71; and Stockel, L., Schwab, M. E., Thoenen, H. (1975). Comparison between the retrograde axonal transport of nerve growth factor and tetanus toxin in motor, sensory and adrenergic neurons. Brain Res. 89, 1-16).
It was found that TH and CGRP can be upregulated by NGF. During normal development, the maximum number of cells in the trigeminal ganglia are found at El3 (see FIG. 1(b). (See Davies, A. M. and A. Lumsden, (1984). Relation of target encounter and neuronal death to nerve growth factor responsiveness in the developing mouse trigeminal ganglion. J. Comp. Neurol. 223, 124-137). These values are twice that measured at postnatal day four, a result consistent with the neurotrophic theory that up to half of all sensory neurons produced during development die as the animal matures. The trigeminal ganglia of mice expressing the K14-NGF transgene had up to double the number of cells than present in control mice.
Since the addition of exogenous NGF is known to block sensory neuron death, the most likely explanation for the increased cell number in the transgenic ganglia is that the supernormal levels of NGF generated by K14NGF transgene expression in the target prevented cell death. Another possibility is that additional neurogenesis was induced by increased levels of NGF. Recent studies have suggested that NGF can act as a mitogen in the anterior pituitary and as a meiotic growth factor during spermatogenesis. (See Borrelli, E., Sawchenko, P. D. Evans, R. M. (1992). Pituitary hyperplasia induced by ectopic expression of nerve growth factor. Proc. Natl. Acad. Sci. U.S.A. 89, 2764-2768; and Parvinen, M., Pelto-Huikko, M., Soder, O., Schults, R., Kaipia, A., Mali, P., Toppari, J., Hakovirta, H., Lonnerberg, P., Ritzen, E. M., Ebendal, T., Olson, L., Hokfelt, T., Persson, H. (1992). Expression of B-nerve growth factor in rat seminiferous epithelium: specific function at the onset of meiosis. J. Cell Biol. 117, 629-642).
However, if NGF does exert a mitogenic effect on peripheral neurons it must occur prior to the first four-to-six weeks of postnatal development (the age at which animals were examined), since we observed no mitotic figures in the trigeminal ganglia despite examination of over 4000 cells/ganglia. In addition, the K14-NGF transgene is active after the period of normal cell birth and at a time when sensory neurons have completed their migration and extended processes into the periphery. Typically, sensory neurons that are this highly differentiated do not undergo neurogenesis. (See Pannese, E. (1974). Adv. Anat. Cell Biol. 47, 1).
In addition to the apparent sensory neuron hyperinnervation, some of the increased innervation resulted from increased survival of NGF responsive sympathetic postganglionic neurons that innervate blood vessels, sweat glands, and the erector pili muscles associated with hair follicles. Sympathetic neuronal cell bodies that innervate the whisker pad skin are located in the superior cervical ganglion, a structure that was also enlarged in the K14-NGF transgenics. The increase in tyrosine hydroxylase immunoreactivity was restricted to the fiber bundles in the superficial portion of the dermis. Blood vessels which normally receive a few intensely stained TH-positive fibers appeared to have the same low level of sympathetic innervation in the transgenic mice. These blood vessels are well removed from the basal keratinocytes expressing the NGF transgene suggesting the hyperinnervation is supported only for those structures near the site of NGF overproduction.
The hyperinnervation of a target may be dependent on factors other than increased expression of NGF. Merkel cells are abundant in vibrissae structures and receive sensory input. In the K14-NGF transgenics, a large increase in the number of CGRP-positive fibers projecting to these cells was found. No TH-positive fibers were seen projecting to these cells even though large numbers of TH-positive axons were seen in nerve bundles immediately adjacent to the vibrissae. These results agree with experiments reported by Edwards et al. in which the rat insulin promotor was utilized to overexpress the NGF cDNA in islet cells of the pancreas. (See Edwards, R. H., Rutter, W. J., Hanahan, D. (1989). Directed expression of NGF to pancreatic β cells in transgenic mice leads to selective hyperinnervation of the islets. Cell 58, 161-170).
Hyperinnervation of the pancreas was observed, though the sole source of innervating fibers appeared to be sympathetic postganglionic neurons. No apparent effect of increased NGF was observed on sensory fibers. From this finding it was proposed that the selective affect on sympathetic axons was due to unidentified cofactors that supported hyperinnervation of the sympathetic but not sensory axons. This type of mechanism may have relevance to another transgenic mouse expression study in which the prolactin promotor was utilized to target NGF expression to the anterior pituitary, a tissue not normally innervated. Though an increased amount of NGF was synthesized, the anterior pituitary was not innervated, i.e., NGF did not induce innervation of an inappropriate target.
The expression paradigm used has allowed an examination of the neurotrophic hypothesis and the effect of increased target-derived NGF on cell survival. The results demonstrate that the increased expression of NGF in the developing epidermis can inhibit, if not completely block, neuron cell death. Moreover, the increase in the number of surviving neurons was independent of changes in the volume of the skin or number of cells in the target tissue (in this case the skin) indicating the trophic factor alone can regulate the number of surviving axons.
These results support the neurotrophic hypothesis and specifically demonstrate that the skin, by regulating the timing and amount of NGF expression independent of innervation plays a central role in the development of the peripheral nervous system.
Construction of K14-NGF transgene and introduction into mice. Plasmid pKS-NGF (a gift from R. Edwards, U.C.L.A.) was cut with Apa 1 (+230) and Pst 1 (+1027) to remove a 797 base pair fragment that encodes the NGF short transcript (Edwards et al., 1989). The overhanging ends of the Apa 1-Pst 1 NGF fragment were removed using mung bean nuclease and the fragment was gel purified and ligated into the BamHl cut, Klenow filled K14-hGH cassette vector (Vassar and Fuchs, 1991, supra). Dr. Elaine Fuchs (University of Chicago) provided the K14-hGH plasmid.
K14-hGH contains 2.1 kbp of 5' upstream sequence of the human K14 keratin gene and a 1.8 kbp intron containing sequence from the human growth hormone (hGH) gene. The hGH sequence serves to upregulate expression of the transgene and provides a polyadenylation signal. (See Sandgren, E. P., Luetteke, N. C., Palmiter, R. D., Brinster, R. L. & Lee, D. C. (1990). Overexpression of TGF alpha in transgenic mice: Induction of epithelial hyperplasia, pancreatic metaplasia, and carcinoma of the breast. Cell, 61, 1210-1135).
The 5-kbp EcoR1 k14-NGF-hGH fragment (FIG. 1a) was isolated on 0.8% Seaplaque agarose gel (FMC Corporation), extracted from the gel using glassmilk purification (Geneclean Bio 101), and run through a NACS column (Bethesda Research Laboratories).
The plasmid used to transfect mouse cells according to the present invention k14-NGF-hGH has been deposited with the American Type Culture Collection under the terms of the Budapest Treaty and is available as Accession No. 69423.
DNA was ethanol precipitated, resuspended in phosphate buffered saline at a concentration of 5 μg/ml, and injected into fertilized C3HB6 mouse (Harlan Laboratory Supplies) embryos. Injections and implantations were carried out using standard procedures. (See Hogan, B., Costantini, F. & Lacy, E. (1986). Manipulating the Mouse Embryo: A Laboratory Manual (Cold Spring Harbor Press, Cold Spring Harbor, N.Y.).
Mice were screened for the transgene using Southern hybridization analysis on DNA extracted from tail or by employing the polymerase chain reaction on DNA samples isolated from ear punches. For Southern hybridizations, 10 ug of DNA was digested with Pst 1, separated on an 0.7% agarose gel, transferred to nitrocellulose by blotting and probed with a random primed 32 P-dCTP (New England Nuclear) labeled probe made to the full length NGF cDNA.
Analysis of mRNA expression by Northern hybridization. Northern analysis was performed on RNA that was isolated from various tissues using guanidium thiocyanate/ phenol/chloroform extraction. (See Chomczynski, P. & Sacchi, N. (1987). Single-step method of RNA isolation by acid guanidinium thiocyanate-phenolchloroform extraction. Analyt. Biochem. 162, 156-159).
Fifteen micrograms of total RNA was resolved on a 1.2% agarose formaldehyde denaturing gel, transferred to Nytran membrane (Schleicher and Schuell) by blotting, baked 2 h in a vacuum oven at 80� C., and then hybridized to a 32 P-dCTP labeled random primed probe made to the full length NGF cDNA.
In situ hybridization. 35 S-labelled cRNA probes were generated by adding lug of linearized pKS-NGF plasmid (linearized with Sac 1 for an antisense probe and EcoR1 for a sense probe) to 2.5mM each ATP, CTP, GTP, 15 mM [35 S]UTP (New England Nuclear) and either T7 or T3 polymerase (Stratagene) to generate antisense and sense probes, respectively. Incubation was for 60 min at 40� C. in transcription buffer containing 50 mM MgCl2, 20 mM spermidine, 20 mM Tris (pH 7.4) and 10 mM DTT. The solution was phenol:chloroformextracted and precipitated with ammonium acetate and 2.5 volumes of ethanol in the presence of 25 μg carrier tRNA.
For in situ hybridization, mouse whisker pad skin was dissected, immediately frozen on dry ice, cryostated at 10-20 μm, mounted onto Vectabond-coated slides (Vector Labs) and stored at -80� C. until hybridized. To hybridize, slides were brought to room temperature, fixed for 10 min in 4% paraformaldehyde, washed in diethyl pyrocarbonate (DEPC)-treated phosphate buffer, transferred to 0.25% acetic anhydride in 0.1 M TEA (pH 8/0) for 10 min at room temperature, dehydrated through a graded series of alcohols and defatted in chloroform. Sections were hybridized for 12-24 hr at 60� C. in a hybridization solution consisting of 1�10TM cpm/ml of 35 -labeled cRNA probe, 50% formamide, 1X Denhardt's solution, 200 mMTris (pH 7.5), 10% dextran sulfate, 0.3 μg/ml salmon sperm DNA, 0.15 mg/ml tRNA, and 40 mM DTT. Slides were washed in 4�SSC (1�SSC=0.15M NaCl, 0.015M sodium citrate, pH 7.0), incubated in 20 mg/ml ribonuclease A (Sigma) dissolved in 10 mM Tris-saline, and then washed through descending concentrations of SSC. The final wash was with 0.1�SSC at 37� C. for 1 hour. Sections were air-dried and placed in X-ray cassettes with Hyperfilm β-Max X-ray film (Amersham). Films were exposed for 3-7s and developed in Kodak D-19. Selected slides were dipped in Kodak NTB-2 liquid emulsion, air-dried and exposed to film. Emulsion dipped slides were developed in D19, fixed in Kodak rapid fixer, counterstained with hematoxylin/eosin or cresyl violet and coverslipped in Permount.
Skin sections from transgenic mice and nontransgenic siblings were processed in parallel. Controls for probe specificity included hybridization using a sense cRNA probe and pretreatment of tissue with RNase. Both of these conditions resulted in the absence of hybridization.
Cell counts of trigeminal neurons. Cell counts were obtained using the method of Coggelshall et al. (See Coggelshall, R. E., Chung, K., Greenwood, D., Hulsebosch, C. (1984). An empirical method for converting nucleolar counts to neuronal numbers. J. Neurosci. Methods 12, 125-132; Coggelshall, R. E., La Forte, R., and Klein, C. M. (1990). Calibration of methods for determining numbers of dorsal root ganglion cells. J. Neurosci. Methods 35, 187-194.).
This method is based on a determination of the number of neuronal nucleoli. Animals were placed under deep anesthesia and perfused transcardially with 4% paraformaldehyde in 0.1M phosphate buffer, pH 7.4. Trigeminal ganglia were removed and immersion fixed in 4% paraformaldehyde, dehydrated in alcohol, defatted and embedded in paraffin. Entire ganglia were serial sectioned at 6 μm and stained with hematoxylin and eosin. Every tenth section was examined at a total magnification of 400� and a drawing tube was used to trace the outline of the section and to demarcate the location of each sensory neuron containing a nucleoli. To compensate for neurons with two or more nucleoli, profiles of randomly selected neurons were reconstructed and the total number of nuclei per 100 neurons was determined. This provided a ration of nucleoli/neuron that was multiplied by the total neurons counted to obtain the total number of neurons per ganglion (Coggeshall et al., 1990, supra).
Immunohistochemistry. Tissue was immersed in fixative for 90 min., transferred to 20-30% sucrose in phosphate buffered saline (PBS) and incubated overnight at 4� C. Samples were cryostated at 10 μm, thaw-mounted onto Vectabond-coated glass slides and processed for immunocytochemistry.
Sections were incubated with primary antibody (diluted in PBS containing 1% normal goat serum and 0.3% Triton X-100), 18-24 h at 4� C. in a humidified chamber, rinsed in PBS for 30 min (3 changes), incubated in a secondary antisera consisting of appropriate fluorophore-conjugated IgG immunoglobulins (diluted in PGC with 0.3% Triton X-100; 1 h at room temperature), rinsed in PBS, and coverslipped.
Primary antibodies recognizing the following proteins were used: anti-150 Kd neurofilament protein (NF 150; 1:300 dilution; Chemicon), calcitonin gene related peptide (CGRP; 1:200 dilution; Chemicon), tyrosine hydroxylase (TH; 1:100 dilution; Chemicon).
Binding of the neurofilament antibody was visualized using anti-rabbit fluorocein isothiocyanate conjugated secondary antibody (Cappel Laboratories). Visualization of CGRP and TH immunoreactivity was achieved using avidin-biotin-peroxidase complex (ABC) kit from Vector Laboratories.
After primary antibody incubation, sections were washed twice in PBS (5 min. each) incubated in biotinylated goat-anti rabbit secondary antibody for 30 min., washed in PBS and incubated in the ABC complex for 30 min. following two PBS washes, immunoreactivity was visualized by incubation in 0.025% 3,3'-diaminobenzidine-4HCl (Aldrich) with 0.05% H2 O2 and 1.5% nickel ammonium sulfate dissolved in 0.1M sodium acetate buffer, pH 6 for 7 minutes.
To test the efficacy of drugs in treating neurodegenerative disorders, a drug candidate is administered to a mouse according to the invention. The behavioral effects and tissue changes of the mouse are determined upon administration of the drug.
FIG. 1 Transgene Construct and characterization of transgenic expression
FIG. 1(a) shows the K14-NGF fusion gene contains the mouse NGF cDNA cloned downstream of the human epidermal K14 gene promoter and enhancer sequences (Vassar et al., 1989). A fragment containing a partial sequence of the hGH gene was ligated downstream of the NGF insert to provide an intron containing sequence and poly A (pA) addition signal. Arrow indicates transcription start site. FIG. 1(b) shows a schematic depicting the onset of NGF expression by E11, and its rise and fall at E15. As the endogenous level of NGF decreases, expression of the K14-NGF transgene begins, rises to a steady level and continues in the adult skin. Actual relative levels of expression are not implied. FIG. 1(c) shows mice were screened for the transgene by Southern hybridization of DNA isolated from tail samples obtained three to four weeks after birth. Blots were probed using a 32 P-labeled DNA sequence complementary to the mouse NGF cDNA. The NGF probe detects both the transgene at 1.8 kbp (arrowhead) and the single copy, endogenous NGF gene (1.58 kbp band below the transgene). Three founder animals were isolated: Lane 1, A-39; Lane 2, A-47; Lane 3, A-56; Lane 4, nontransgenic control.
FIG. 2 Expression of the K14-NGF transgene is tissue specific
FIG. 2(a) examines the expression level of the K14-NGF transgene for each line. RNA was isolated from full thickness skin of 4-6 week-old F1 mice and analyzed using Northern hybridization. Ten micrograms of total RNA was resolved on a formaldehyde denaturing gel, transferred to Nytran membrane and probed with a 32 P-labeled DNA sequence complementary to the NGFmRNA. All samples were loaded onto a single gel. Two film exposure were done in order to detect the low level expression of endogenous NGF transcript. Shown is a 3 d exposure, FIG. 2(a) and a 10 d exposure, FIG. 2(b), of the same filter. Lane 1, skin RNA of nontransgenic control. A faint band is visible at 1.3 kb corresponding to the endogenous NGF transcript; lane 2, RNA from tail skin of A-39 founder. Transgene mRNA band (arrowhead) is apparent at the predicted size of 1.9 kb; lane 3, skin RNA of A-47 F1, lane 4, skin RNA of A-56 F1, lane 5, skin RNA of A-47 F1 (loaded twice); lane 6, tongue RNA of A-56 F1. Tongue is a keratinized, K14 keratin expressing structure expected to have transgene expression.
FIG. 2(b) examines the tissue specificity of K14-NGF expression. RNA from brain (lane 7, A-56; lane 13, control), heart (lane 8, A-56; lane 14, control), liver (lane 9, A-56, lane 15, control), and kidney (lane 10, A-56; lane 16, control) were tested. Transgene expression was not evident in these structures, though the 1.3-kb endogenous NGF transcript could be detected in all but liver samples. Lane 11, control skin RNA; lane 12, control tongue RNA. The 28s and 18s ribosomal RNA size markers are indicated by bars on right.
FIG. 3 K14-NGF transgene produces elevated levels of NGF mRNA in skin.
FIG. 3 shows cellular expression of the K14-NGF transgene in skin from A-56 F1 transgenics was examined by in situ hybridization. 35 S-labeled cRNA sense and antisense probes were hybridized to frozen sections of whisker pad skin. Basal cells (arrows) in whisker pad epidermis, FIG. 3(a), and vibrissa, FIG. 3(c), of control animals had silver grain density at background levels. A-56 F1 transgenic mice had a greater labeling density of whisker pad basal cells, FIG. 3(b) (arrows), and vibrissa, FIG. 3(d). Control hybridizations using a sense riproprobe showed background levels of hybridization. Bar=50 μm.
FIG. 4 Expression of K14-NGF causes hyperinnervation of skin.
Overproduction of NGF by the epidermis caused by hyperinnervation that was evident with normal histology. Skin from dorsum, (FIGS. 4a and 4b) and whisker pad (FIG. 4c and 4d) was processed for paraffin histology and sections stained with hematoxylin and eosin. Control skin (FIG. 4a and 4c) had few apparent neuron bundles whereas A-56 F1 transgenic skin (FIG. 4b and 4d) showed numerous, enlarged fiber bundles (arrowheads. * =muscle. Bar=10 μm.
FIG. 5 Immunolabeling of transgenic skin reveals a hyperinnervation of sensory and sympathetic neurons.
To verify that the fiber bundles in the skin were composed of neuronal processes, frozen sections of whisker pad skin from control (FIG. 5a) and A-56 F1 transgenics (FIG. 5b) were immunolabeled with an antineurofilament 150 antibody followed by incubation with a goat anti-rabbit FITC conjugated secondary. Control skin had immunoreactive fibers (arrowheads) in the reticular layer of the dermis, deep dermis, and around hair follicles and vibrissa (arrow). A significant increase in the number of fibers and bundles was seen in an equivalent region of A-56 transgenic skin (FIG. 5b). To determine whether the large nerve bundles in the A-56 skin contained sympathetic fibers, sections were immunolabeled with an anti-TH antibody followed by a biotinylated goat-anti rabbit secondary processing with DAB (FIG. 5c). TH positive fibers, which were rare in control skin, were found throughout the dermis (arrowheads). To examine peripheral sensory fibers in the transgenic skin, A-56 F1 transgenics were labeled with anti-CGRP followed by a biotinylated goat-anti rabbit secondary antibody (FIG. 5d). Large numbers of CGRP positive fibers and nerve bundles were present in the dermis and around vibrissa. Inset shows vibrissa surrounded by CGRP positive fibers (arrowheads). Bar=5 μm.
FIG. 6 Expression of K14-NGF causes enlargement of the trigeminal and superior cervical ganglia.
Trigeminal ganglia (FIG. 6a) and superior cervical ganglia (FIG. 6b) were removed from control A-48 F1, and A-56 F1 mice. For both the trigeminal and sympathetic superior cervical ganglia, controls (left) were smaller than either the A-48 (center) or A-56 ganglia (right. Bar=1 mm).
The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without departing from the generic concept and therefore such adaptations are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description only and not of limitation. It is obvious that certain changes and modifications may be practiced within the scope of the appended claims.
|Cited Patent
|Filing date
|Publication date
|Nov 23, 1977
|Jan 22, 1980
|The Massachusetts General Hospital
|Nerve growth factor
|Jun 22, 1984
|Apr 12, 1988
|President And Fellows Of Harvard College
|Transgenic non-human mammals
|Aug 30, 1988
|Jan 21, 1992
|The General Hospital Corporation
|Recombinant human nerve growth factor
|Feb 17, 1989
|Dec 29, 1992
|President And Fellows Of Harvard College
|Animal model for benign prostatic disease
|Dec 5, 1988
|Dec 29, 1992
|Genpharm International
|Transgenic mice depleted in mature t-cells and methods for making transgenic mice
|Sep 3, 1987
|Dec 29, 1992
|Ohio University/Edison Animal Biotechnolgy Center
|Virus-resistant transgenic mice
|Aug 30, 1989
|Jan 19, 1993
|Barde Yves Alain
|Brain-derived neurotrophic factor
|Mar 14, 1991
|Jul 27, 1993
|The United States Of America As Represented By The Department Of Health And Human Services
|Trk tyrosine kinase receptor is the physiological receptor for nerve growth factor
|Apr 23, 1992
|Jan 21, 1993
|Regeneron Pharmaceuticals, Inc.
|Method and assay system for neurotrophin activity
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|Citing Patent
|Filing date
|Publication date
|Apr 9, 1998
|Aug 21, 2001
|Genentech, Inc.
|Method of dopaminergic and serotonergic neuron formation from neuroprogenitor cells
|Jul 17, 1998
|May 3, 2005
|Amanda Reynolds-Jahoda
|Gene therapy vehicle comprising dermal sheath tissue
|Dec 14, 2001
|Jul 25, 2006
|The Regents Of The University Of California
|Gene-targeted animal model of apolipoprotein E4 domain interaction and uses thereof
|Jul 12, 2005
|Jun 28, 2011
|Van Andel Research Institute
|Immune-compromised transgenic mice expressing human hepatocyte growth factor (hHGF)
|Jun 7, 2006
|Oct 12, 2006
|Weisgraber Karl H
|Gene-targeted animal model of apolipoprotein E4 domain interaction and uses thereof
|Jul 12, 2005
|Aug 14, 2008
|Van Andel Research Institute
|Immune-Compromised Transgenic Mice Expressing Human Hepatocyte Growth Factor (Hhgf)
|Jun 10, 1998
|Dec 17, 1998
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|U.S. Classification
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|International Classification
|Cooperative Classification
|European Classification
|Apr 9, 1996
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Mister Bed City Hotel Bagnolet: Find photos, reviews, information, amenities and compare prices for 2 stars hotels in Bagnolet, France.
Mister Bed City Hotel Bagnolet provides a modern setting when in Bagnolet. It also offers a 24-hour reception, luggage storage and a hair salon.
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Mister Bed City Hotel Bagnolet provides spacious rooms equipped with a flat-screen TV, a shower and cable/satellite channels. Also available are a telephone, wireless internet access and a private bathroom.
Mister Bed City serves breakfast each morning, and has a bar where guests can unwind with a drink.
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Hotel features
Cable / Satellite TV, TV, Shower, En suite, Private Bathroom
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Showing 1-2 of 2 Romantic Resorts
Resort Properties of Angel Fire
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Escape for a weekend for two into the great wide open of New Mexico! Escape the busy city life and relax with your sweetheart as you get back to what's really important, togetherness. Inquire today! ... more
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Glendale Guesthouse Bolton: Find photos, reviews, information, amenities and compare prices for 3 stars hotels in Bolton, United Kingdom.
Less than a 20-minute walk from Bolton Bus Station, Glendale provides guests with a convenient base when visiting Bolton. It is ideally located for guests wanting to visit local attractions.
Hotel description
This 3-star guest house offers a 24-hour reception, a garden and parking. Wi-Fi is also provided.
Glendale Guesthouse Bolton provides spacious rooms equipped with tea and coffee making facilities. Guests staying in the rooms also have access to a shared bathroom.
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Users who viewed Glendale Guesthouse Bolton also viewed these 3 stars Bolton hotels:
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WiFi is available in public areas and costs EUR 8 per 24 hours.
Free parking! Free public parking is possible on site (reservation is not needed).
From 16:00 Until 20:00 hours
From 07:00 Until 09:00 hours
All children are welcome.All children under 2 years are charged EUR 5 per night for children's cots/cribs.All children under 12 years are charged EUR 15 per night for extra beds.All further older children or adults are charged EUR 15 per night for extra beds.The maximum number of extra beds in a room is 2.
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Located in Torre Lapillo, this air-conditioned holiday home is situated 29 km from Lecce. The unit is 28 km from Gallipoli.
Gallipoli is 28 km from Casa Toto' SX 1P, while Brindisi is 41 km from the property. Brindisi - Salento Airport is 43 km away.
Brindisi is 41 km from Altomare Case Vacanza, while San Cassiano is 49 km away. The nearest airport is Brindisi - Salento Airport, 43 km from the property.
Featuring free WiFi throughout the property, Hotel La Spiaggia offers accommodation in Torre Lapillo. The hotel has a sun terrace and views of the sea, and guests can enjoy a...
Casa Vacanza Torre Lapillo is a holiday home located in Torre Lapillo, 29 km from Lecce. The air-conditioned unit is 28 km from Gallipoli.
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Here's the 120ml Ru Kiln moon white cup I bought from DTH
And here it is a month after use.
Zensuji wrote:Mine was like that after about 2 weeks.
What I did was drink tea from it all day everyday. Mostly green oolongs and didnt wash it till the end of the day. So about 10 refills a day and no washing till just before bed.
Its beautiful. I love staring into it. Some of the crazing runs from one side of the cup to the other in a huge sweeping S.
And yeah IPT is bang on the money. Its the repeated tea and then dry cycle that gets it going. You could probably get it like mine in a matter of days if you tried. I think mines reached its max but maybe I'll take another picture in a months time and we'll see.
IPT wrote:Soaking it won't really help. The best way to speed it up is to fill it with heavily brewed Shupu Tea and pour it out. Let the cup dry and repeat. Getting it wet and drying it out will do it quickly.
If you soak it, or let it dry out, the crackles will seem stained, but once you put hot tea in it, the darkening will disappear.
People ask me to help them darken their ru kiln ware, so I've done it quite a bit.
IPT wrote:Of course, the best way to stain it is to just use it. I get a sense of pride from mine because I know I did it cup by cup.
woozl wrote:Mrs. Woozie has celadon going the wood fire this weekend...


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Hedge Funds
Stark Investments closes European convertibles fund
Wisconsin-based Stark Investments has liquidated its Stark Europe Fund, citing a lack of opportunities in European convertibles. Most of the money has apparently been switched into the firm's big global multi-strategy vehicle, Shepherd Investments.
The $35 million fund was always just a small offshoot of the $4 billion shop's multi-strategy product range.
The decision to shut down the fund continues a trend that has already seen various other managers, including Axis Capital and Gartmore, shut down convertible strategies in Europe this year.
Stark continues to run two other geographically-focused funds that focus on small sub-sectors of its huge multi-strategy master


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NDPDD head arrested
State and local authorities swooped in and arrested the longtime executive director of North Delta Planning and Development District, Glenn K. Brown, Thursday morning (July 1) at his office in Batesville.
Brown, 49, was booked at the Panola County jail at 8:25 and charged with fraud, according to the jail log.
Despite Brown's arrest, employees of North Delta Planning are still at work and performing their jobs, said (Panola) Board of Supervisors president Gary Thompson.
"The doors are still open," he said.
Latest News
Delivering toys and smiles Sheriff K.C. Hamp, Sr....
Local History Yazoo Pass, Part 3: Yankees reach the Coldwater This week continues a...
Progress in local schools recognized Citing a "laser...
Local schools getting recognition from state ceremonies today Tunica is one of...
White Oak to get natural gas lines The cost of...
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Beigelman, Avraham; Bacharier, Leonard B.
Propose of review: Cumulative evidence suggest that early life bronchiolitis is a major risk factor for subsequent wheezing episodes and asthma. The purpose of this review is to present the recent findings and current perspectives regarding the interplay between bronchiolitis and long-term respiratory outcomes.
Recent findings: Recent studies have supported the long-recognized link between early life severe respiratory syncytial virus bronchiolitis and the physician diagnosis of asthma by school age, and this association appears to continue into early adulthood. Evidence is accumulating regarding the role of early life infection with human rhinovirus as an important antecedent for future asthma. Whether viral bronchiolitis is causal or an early manifestation of future asthma remains uncertain. Vitamin D status has emerged as a potential modifying factor for viral-induced wheeze and could potentially influence the development of asthma.
Summary: Viral bronchiolitis early in life is a major and potential long-term risk factor for subsequent wheezing and asthma. Whether the association between bronchiolitis and subsequent asthma is due to causality or a reflection of predisposition may be dependent on host factors and virus-specific effects.
Copyright © 2013 Wolters Kluwer Health, Inc. All rights reserved.
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Nets of Wonder
Genuine and intellectual, Nets of Wonder sojourns into a refreshing, realistic gay love story. In Nets of Wonder, author Robert Heylmun traces the romantic life of Will, a gay... Read More
Genuine and intellectual, Nets of Wonder sojourns into a refreshing, realistic gay love story. In Nets of Wonder, author Robert Heylmun traces the romantic life of Will, a gay... Read More
Deanna Kahler invites readers to get into the spirit of love with this lyrical work. Celeste loves Connor, but Connor is dead. Are the gifts she receives—a pink rose, a little... Read More
The definition of love itself is an underlying social issue explored in depth, though not addressed in a typically overt manner, throughout this touching story. The exciting... Read More
Suspense and romance bolster the appeal of this engaging tale of danger, forgiveness, and love. Two gunshots change Emma Potter's world forever in Ayla Wood's debut novel,... Read More
Lush narrative, intellectual conversation, and emotional introspection help us experience an exotic locale through the eyes of its people. In a weighty tome that spans more than... Read More
Mesmerizing and meticulous, David Bradford examines the emotional side of intimacy. In this thoughtful, soul-searching romance, a woman distinguishes between loyalty and love.... Read More
This debut work is a clear-eyed look at the many ways plans can derail, and at the resilience of those who get back on track. Four men, four women, one college semester in... Read More
Here's a pleasant surprise: a romance with admirable, realistic characters. From the author of three previous books about the triumph of the human spirit comes a tale of... Read More
Mystery, suspense, romance, adventure, and cricket make an intriguing mix. In Cricket Maiden, Algenes Edmund Dantes presents a story of suspense, mystery, and romance centered... Read More
Gripping and disturbing, this page-turner featuring a courageous spy heroine during WWII defies genre. A bilingual woman in the British Women's Army sneaks into Germany during... Read More
Ryan expertly weaves dialogue and description in this continuing story of a woman and her obsession with an ancestral home. Dot Ryan once again proves her abilities as an... Read More
In this tightly-written war thriller, the reader is brought into the scene quickly as the author introduces the characters in situ and one by one—characters whose fates will... Read More


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Ways To Finding Investors For Your Startup – Beginners Guide
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Microloans For Business Funding
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Finding A Guarantee For A Private Loan
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Hearing The Warning Bells Of The Bond Market
The financial news media love the drama of the stock market, often at the expense of equally important financial news — the bond market. True followers of the financial markets and smart economic analysts know there is much more than
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Coming up with a down payment can definitely be the most difficult part of affording to buy a new house especially if this is the first home you are embarking on purchasing. However, there are a variety of ways in
Short Selling – Its Meaning in the Trade of Stocks and Mortgages
In recent years, short sales have become part of daily vernacular. For many people they know it to be a process where some item, usually a house, is sold for much less than what is owed on it. After
How Private Equity Investment Works
Private equity is a type of capital which institutions and high-worth individual use to invest in and acquire equity ownership in certain companies. Private equity firms help raise funds and manage these types of monies as they work to provide
Ways To Finding Investors For Your Startup – Beginners Guide
Introduction Starting a new business is an exciting time for many business owners. However, according to research, it has showed that too often new business ventures are unable to succeed due to lack of capital in the first two years