The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.
20 Related JoVE Articles!
Detection of Neuritic Plaques in Alzheimer's Disease Mouse Model
Institutions: The University of British Columbia.
Alzheimer's disease (AD) is the most common neurodegenerative disorder leading to dementia. Neuritic plaque formation is one of the pathological hallmarks of Alzheimer's disease. The central component of neuritic plaques is a small filamentous protein called amyloid β protein (Aβ)1
, which is derived from sequential proteolytic cleavage of the beta-amyloid precursor protein (APP) by β-secretase and γ-secretase. The amyloid hypothesis entails that Aγ-containing plaques as the underlying toxic mechanism in AD pathology2
. The postmortem analysis of the presence of neuritic plaque confirms the diagnosis of AD. To further our understanding of Aγ neurobiology in AD pathogenesis, various mouse strains expressing AD-related mutations in the human APP genes were generated. Depending on the severity of the disease, these mice will develop neuritic plaques at different ages. These mice serve as invaluable tools for studying the pathogenesis and drug development that could affect the APP processing pathway and neuritic plaque formation. In this protocol, we employ an immunohistochemical method for specific detection of neuritic plaques in AD model mice. We will specifically discuss the preparation from extracting the half brain, paraformaldehyde fixation, cryosectioning, and two methods to detect neurotic plaques in AD transgenic mice: immunohistochemical detection using the ABC and DAB method and fluorescent detection using thiofalvin S staining method.
Neuroscience, Issue 53, Alzheimer’s disease, neuritic plaques, Amyloid β protein, APP, transgenic mouse
Identification of Sleeping Beauty Transposon Insertions in Solid Tumors using Linker-mediated PCR
Institutions: University of Minnesota, Minneapolis, University of Minnesota, Minneapolis.
Genomic, proteomic, transcriptomic, and epigenomic analyses of human tumors indicate that there are thousands of anomalies within each cancer genome compared to matched normal tissue. Based on these analyses it is evident that there are many undiscovered genetic drivers of cancer1
. Unfortunately these drivers are hidden within a much larger number of passenger anomalies in the genome that do not directly contribute to tumor formation. Another aspect of the cancer genome is that there is considerable genetic heterogeneity within similar tumor types. Each tumor can harbor different mutations that provide a selective advantage for tumor formation2
. Performing an unbiased forward genetic screen in mice provides the tools to generate tumors and analyze their genetic composition, while reducing the background of passenger mutations. The Sleeping Beauty
(SB) transposon system is one such method3
. The SB system utilizes mobile vectors (transposons) that can be inserted throughout the genome by the transposase enzyme. Mutations are limited to a specific cell type through the use of a conditional transposase allele that is activated by Cre Recombinase
. Many mouse lines exist that express Cre Recombinase
in specific tissues. By crossing one of these lines to the conditional transposase allele (e.g.
Lox-stop-Lox-SB11), the SB system is activated only in cells that express Cre Recombinase
. The Cre Recombinase
will excise a stop cassette that blocks expression of the transposase allele, thereby activating transposon mutagenesis within the designated cell type. An SB screen is initiated by breeding three strains of transgenic mice so that the experimental mice carry a conditional transposase allele, a concatamer of transposons, and a tissue-specific Cre Recombinase
allele. These mice are allowed to age until tumors form and they become moribund. The mice are then necropsied and genomic DNA is isolated from the tumors. Next, the genomic DNA is subjected to linker-mediated-PCR (LM-PCR) that results in amplification of genomic loci containing an SB transposon. LM-PCR performed on a single tumor will result in hundreds of distinct amplicons representing the hundreds of genomic loci containing transposon insertions in a single tumor4
. The transposon insertions in all tumors are analyzed and common insertion sites (CISs) are identified using an appropriate statistical method5
. Genes within the CIS are highly likely to be oncogenes or tumor suppressor genes, and are considered candidate cancer genes. The advantages of using the SB system to identify candidate cancer genes are: 1) the transposon can easily be located in the genome because its sequence is known, 2) transposition can be directed to almost any cell type and 3) the transposon is capable of introducing both gain- and loss-of-function mutations6
. The following protocol describes how to devise and execute a forward genetic screen using the SB transposon system to identify candidate cancer genes (Figure 1
Genetics, Issue 72, Medicine, Cancer Biology, Biomedical Engineering, Genomics, Mice, Genetic Techniques, life sciences, animal models, Neoplasms, Genetic Phenomena, Forward genetic screen, cancer drivers, mouse models, oncogenes, tumor suppressor genes, Sleeping Beauty transposons, insertions, DNA, PCR, animal model
Dissection of Saccharomyces Cerevisiae Asci
Institutions: Concordia University.
Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae
exists in either a haploid or diploid state. The ability to combine alleles from two haploids and the ability to introduce modifications to the genome requires the production and dissection of asci. Asci production from haploid cells begins with the mating of two yeast haploid strains with compatible mating types to produce a diploid strain. This can be accomplished in a number of ways either on solid medium or in liquid. It is advantageous to select for the diploids in medium that selectively promotes their growth compared to either of the haploid strains. The diploids are then allowed to sporulate on nutrient-poor medium to form asci, a bundle of four haploid daughter cells resulting from meiotic reproduction of the diploid. A mixture of vegetative cells and asci is then treated with the enzyme zymolyase to digest away the membrane sac surrounding the ascospores of the asci. Using micromanipulation with a microneedle under a dissection microscope one can pick up individual asci and separate and relocate the four ascopores. Dissected asci are grown for several days and tested for the markers or alleles of interest by replica plating onto appropriate selective media.
Cellular Biology, Issue 27, asci, ascospores, diploid, zygote, sporulation, yeast dissection, micromanipulator
Spheroid Assay to Measure TGF-β-induced Invasion
Institutions: Leiden University Medical Centre.
TGF-β has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis at later stage1,2
. Moreover, TGF-β is frequently overexpressed in breast cancer and its expression correlates with poor prognosis and metastasis 3,4
. The mechanisms by which TGF-β induces invasion are not well understood.
TGF-β elicits its cellular responses via TGF-β type II (TβRII) and type I (TβRI) receptors. Upon TGF-β-induced heteromeric complex formation, TβRII phosphorylates the TβRI. The activated TβRI initiates its intracellular canonical signaling pathway by phosphorylating receptor Smads (R-Smads), i.e. Smad2 and Smad3. These activated R-Smads form heteromeric complexes with Smad4, which accumulate in the nucleus and regulate the transcription of target genes5
. In addition to the previously described Smad pathway, receptor activation results in activation of several other non-Smad signaling pathways, for example Mitogen Activated Protein Kinase (MAPK) pathways6
To study the role of TGF-β in different stages of breast cancer, we made use of the MCF10A cell system. This system consists of spontaneously immortalized MCF10A1 (M1) breast epithelial cells7
, the H-RAS transformed M1-derivative MCF10AneoT (M2), which produces premalignant lesions in mice8
, and the M2-derivative MCF10CA1a (M4), which was established from M2 xenografts and forms high grade carcinomas with the ability to metastasize to the lung9
. This MCF10A series offers the possibility to study the responses of cells with different grades of malignancy that are not biased by a different genetic background.
For the analysis of TGF-β-induced invasion, we generated homotypic MCF10A spheroid cell cultures embedded in a 3D collagen matrix in vitro
(Fig 1). Such models closely resemble human tumors in vivo
by establishing a gradient of oxygen and nutrients, resulting in active and invasive cells on the outside and quiescent or even necrotic cells in the inside of the spheroid10
. Spheroid based assays have also been shown to better recapitulate drug resistance than monolayer cultures11
. This MCF10 3D model system allowed us to investigate the impact of TGF-β signaling on the invasive properties of breast cells in different stages of malignancy.
Medicine, Issue 57, TGF-β, TGF, breast cancer, assay, invasion, collagen, spheroids, oncology
Isolation of Cellular Lipid Droplets: Two Purification Techniques Starting from Yeast Cells and Human Placentas
Institutions: University of Tennessee, University of Tennessee.
Lipid droplets are dynamic organelles that can be found in most eukaryotic and certain prokaryotic cells. Structurally, the droplets consist of a core of neutral lipids surrounded by a phospholipid monolayer. One of the most useful techniques in determining the cellular roles of droplets has been proteomic identification of bound proteins, which can be isolated along with the droplets. Here, two methods are described to isolate lipid droplets and their bound proteins from two wide-ranging eukaryotes: fission yeast and human placental villous cells. Although both techniques have differences, the main method - density gradient centrifugation - is shared by both preparations. This shows the wide applicability of the presented droplet isolation techniques.
In the first protocol, yeast cells are converted into spheroplasts by enzymatic digestion of their cell walls. The resulting spheroplasts are then gently lysed in a loose-fitting homogenizer. Ficoll is added to the lysate to provide a density gradient, and the mixture is centrifuged three times. After the first spin, the lipid droplets are localized to the white-colored floating layer of the centrifuge tubes along with the endoplasmic reticulum (ER), the plasma membrane, and vacuoles. Two subsequent spins are used to remove these other three organelles. The result is a layer that has only droplets and bound proteins.
In the second protocol, placental villous cells are isolated from human term placentas by enzymatic digestion with trypsin and DNase I. The cells are homogenized in a loose-fitting homogenizer. Low-speed and medium-speed centrifugation steps are used to remove unbroken cells, cellular debris, nuclei, and mitochondria. Sucrose is added to the homogenate to provide a density gradient and the mixture is centrifuged to separate the lipid droplets from the other cellular fractions.
The purity of the lipid droplets in both protocols is confirmed by Western Blot analysis. The droplet fractions from both preps are suitable for subsequent proteomic and lipidomic analysis.
Bioengineering, Issue 86, Lipid droplet, lipid body, fat body, oil body, Yeast, placenta, placental villous cells, isolation, purification, density gradient centrifugation
Competitive Genomic Screens of Barcoded Yeast Libraries
Institutions: University of Toronto, University of Toronto, University of Toronto, National Human Genome Research Institute, NIH, Stanford University , University of Toronto.
By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli
have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans
barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.
Biochemistry, Issue 54, chemical biology, chemogenomics, chemical probes, barcode microarray, next generation sequencing
Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization Strategies
Institutions: University of California, Los Angeles .
In the biological sciences there have been technological advances that catapult the discipline into golden ages of discovery. For example, the field of microbiology was transformed with the advent of Anton van Leeuwenhoek's microscope, which allowed scientists to visualize prokaryotes for the first time. The development of the polymerase chain reaction (PCR) is one of those innovations that changed the course of molecular science with its impact spanning countless subdisciplines in biology. The theoretical process was outlined by Keppe and coworkers in 1971; however, it was another 14 years until the complete PCR procedure was described and experimentally applied by Kary Mullis while at Cetus Corporation in 1985. Automation and refinement of this technique progressed with the introduction of a thermal stable DNA polymerase from the bacterium Thermus aquaticus
, consequently the name Taq
PCR is a powerful amplification technique that can generate an ample supply of a specific segment of DNA (i.e., an amplicon) from only a small amount of starting material (i.e., DNA template or target sequence). While straightforward and generally trouble-free, there are pitfalls that complicate the reaction producing spurious results. When PCR fails it can lead to many non-specific DNA products of varying sizes that appear as a ladder or smear of bands on agarose gels. Sometimes no products form at all. Another potential problem occurs when mutations are unintentionally introduced in the amplicons, resulting in a heterogeneous population of PCR products. PCR failures can become frustrating unless patience and careful troubleshooting are employed to sort out and solve the problem(s). This protocol outlines the basic principles of PCR, provides a methodology that will result in amplification of most target sequences, and presents strategies for optimizing a reaction. By following this PCR guide, students should be able to:
● Set up reactions and thermal cycling conditions for a conventional PCR experiment
● Understand the function of various reaction components and their overall effect on a PCR experiment
● Design and optimize a PCR experiment for any DNA template
● Troubleshoot failed PCR experiments
Basic Protocols, Issue 63, PCR, optimization, primer design, melting temperature, Tm, troubleshooting, additives, enhancers, template DNA quantification, thermal cycler, molecular biology, genetics
Generation of Enterobacter sp. YSU Auxotrophs Using Transposon Mutagenesis
Institutions: Youngstown State University.
Prototrophic bacteria grow on M-9 minimal salts medium supplemented with glucose (M-9 medium), which is used as a carbon and energy source. Auxotrophs can be generated using a transposome. The commercially available, Tn5
-derived transposome used in this protocol consists of a linear segment of DNA containing an R6Kγ
replication origin, a gene for kanamycin resistance and two mosaic sequence ends, which serve as transposase binding sites. The transposome, provided as a DNA/transposase protein complex, is introduced by electroporation into the prototrophic strain, Enterobacter
sp. YSU, and randomly incorporates itself into this host’s genome. Transformants are replica plated onto Luria-Bertani agar plates containing kanamycin, (LB-kan) and onto M-9 medium agar plates containing kanamycin (M-9-kan). The transformants that grow on LB-kan plates but not on M-9-kan plates are considered to be auxotrophs. Purified genomic DNA from an auxotroph is partially digested, ligated and transformed into a pir+ Escherichia coli
) strain. The R6Kγ
replication origin allows the plasmid to replicate in pir+ E. coli
strains, and the kanamycin resistance marker allows for plasmid selection. Each transformant possesses a new plasmid containing the transposon flanked by the interrupted chromosomal region. Sanger sequencing and the Basic Local Alignment Search Tool (BLAST) suggest a putative identity of the interrupted gene. There are three advantages to using this transposome mutagenesis strategy. First, it does not rely on the expression of a transposase gene by the host. Second, the transposome is introduced into the target host by electroporation, rather than by conjugation or by transduction and therefore is more efficient. Third, the R6Kγ
replication origin makes it easy to identify the mutated gene which is partially recovered in a recombinant plasmid. This technique can be used to investigate the genes involved in other characteristics of Enterobacter
sp. YSU or of a wider variety of bacterial strains.
Microbiology, Issue 92, Auxotroph, transposome, transposon, mutagenesis, replica plating, glucose minimal medium, complex medium, Enterobacter
A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses
Institutions: Emory University, Emory University.
The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target conserved portions of the HIV-1 genome that escape with difficulty. Sequence changes attributed to cellular immune pressure arise across the genome during infection, and if found within conserved regions of the genome such as Gag, can affect the ability of the virus to replicate in vitro
. Transmission of HLA-linked polymorphisms in Gag to HLA-mismatched recipients has been associated with reduced set point viral loads. We hypothesized this may be due to a reduced replication capacity of the virus. Here we present a novel method for assessing the in vitro
replication of HIV-1 as influenced by the gag
gene isolated from acute time points from subtype C infected Zambians. This method uses restriction enzyme based cloning to insert the gag
gene into a common subtype C HIV-1 proviral backbone, MJ4. This makes it more appropriate to the study of subtype C sequences than previous recombination based methods that have assessed the in vitro
replication of chronically derived gag-pro
sequences. Nevertheless, the protocol could be readily modified for studies of viruses from other subtypes. Moreover, this protocol details a robust and reproducible method for assessing the replication capacity of the Gag-MJ4 chimeric viruses on a CEM-based T cell line. This method was utilized for the study of Gag-MJ4 chimeric viruses derived from 149 subtype C acutely infected Zambians, and has allowed for the identification of residues in Gag that affect replication. More importantly, the implementation of this technique has facilitated a deeper understanding of how viral replication defines parameters of early HIV-1 pathogenesis such as set point viral load and longitudinal CD4+ T cell decline.
Infectious Diseases, Issue 90, HIV-1, Gag, viral replication, replication capacity, viral fitness, MJ4, CEM, GXR25
A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos
Institutions: University of Notre Dame.
Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ
hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.
Developmental Biology, Issue 93, zebrafish, chemical genetics, chemical screen, in vivo small molecule screen, drug discovery, whole mount in situ hybridization (WISH), high-throughput screening (HTS), high-content screening (HCS)
Morris Water Maze Test for Learning and Memory Deficits in Alzheimer's Disease Model Mice
Institutions: University of British Columbia.
The Morris Water Maze (MWM) was first established by neuroscientist Richard G. Morris in 1981 in order to test hippocampal-dependent learning, including acquisition of spatial memoryand long-term spatial memory 1
. The MWM is a relatively simple procedure typically consisting of six day trials, the main advantage being the differentiation between the spatial (hidden-platform) and non-spatial (visible platform) conditions 2-4
. In addition, the MWM testing environment reduces odor trail interference 5
. This has led the task to be used extensively in the study of the neurobiology and neuropharmacology of spatial learning and memory. The MWM plays an important role in the validation of rodent models for neurocognitive disorders such as Alzheimer’s Disease 6, 7
. In this protocol we discussed the typical procedure of MWM for testing learning and memory and data analysis commonly used in Alzheimer’s disease transgenic model mice.
Neuroscience, Issue 53, Morris Water Maze, spatial memory testing, hippocampal dependent learning, Alzheimer's Disease
Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood
Institutions: U.T. MD Anderson Cancer Center, U.T. MD Anderson Cancer Center.
The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer a biologic product with the potential for superior (i) recognition of tumor-associated antigens (TAAs), (ii) persistence after infusion, (iii) potential for migration to tumor sites, and (iv) ability to recycle effector functions within the tumor microenvironment. Most approaches to genetic manipulation of T cells engineered for human application have used retrovirus and lentivirus for the stable expression of CAR1-3
. This approach, although compliant with current good manufacturing practice (GMP), can be expensive as it relies on the manufacture and release of clinical-grade recombinant virus from a limited number of production facilities. The electro-transfer of nonviral plasmids is an appealing alternative to transduction since DNA species can be produced to clinical grade at approximately 1/10th
the cost of recombinant GMP-grade virus. To improve the efficiency of integration we adapted Sleeping Beauty
(SB) transposon and transposase for human application4-8
. Our SB system uses two DNA plasmids that consist of a transposon coding for a gene of interest (e.g.
generation CD19-specific CAR transgene, designated CD19RCD28) and a transposase (e.g.
SB11) which inserts the transgene into TA dinucleotide repeats9-11
. To generate clinically-sufficient numbers of genetically modified T cells we use K562-derived artificial antigen presenting cells (aAPC) (clone #4) modified to express a TAA (e.g.
CD19) as well as the T cell costimulatory molecules CD86, CD137L, a membrane-bound version of interleukin (IL)-15 (peptide fused to modified IgG4 Fc region) and CD64 (Fc-γ receptor 1) for the loading of monoclonal antibodies (mAb)12
. In this report, we demonstrate the procedures that can be undertaken in compliance with cGMP to generate CD19-specific CAR+
T cells suitable for human application. This was achieved by the synchronous electro-transfer of two DNA plasmids, a SB transposon (CD19RCD28) and a SB transposase (SB11) followed by retrieval of stable integrants by the every-7-day additions (stimulation cycle) of γ-irradiated aAPC (clone #4) in the presence of soluble recombinant human IL-2 and IL-2113
. Typically 4 cycles (28 days of continuous culture) are undertaken to generate clinically-appealing numbers of T cells that stably express the CAR. This methodology to manufacturing clinical-grade CD19-specific T cells can be applied to T cells derived from peripheral blood (PB) or umbilical cord blood (UCB). Furthermore, this approach can be harnessed to generate T cells to diverse tumor types by pairing the specificity of the introduced CAR with expression of the TAA, recognized by the CAR, on the aAPC.
Immunology, Issue 72, Cellular Biology, Medicine, Molecular Biology, Cancer Biology, Biomedical Engineering, Hematology, Biochemistry, Genetics, T-Lymphocytes, Antigen-Presenting Cells, Leukemia, Lymphoid, Lymphoma, Antigens, CD19, Immunotherapy, Adoptive, Electroporation, Genetic Engineering, Gene Therapy, Sleeping Beauty, CD19, T cells, Chimeric Antigen Receptor, Artificial Antigen Presenting Cells, Clinical Trial, Peripheral Blood, Umbilical Cord Blood, Cryopreservation, Electroporation
The Green Monster Process for the Generation of Yeast Strains Carrying Multiple Gene Deletions
Institutions: J. Craig Venter Institute, J. Craig Venter Institute, University of Toronto, Mt Sinai Hospital.
Phenotypes for a gene deletion are often revealed only when the mutation is tested in a particular genetic background or environmental condition1,2
. There are examples where many genes need to be deleted to unmask hidden gene functions3,4
. Despite the potential for important discoveries, genetic interactions involving three or more genes are largely unexplored. Exhaustive searches of multi-mutant interactions would be impractical due to the sheer number of possible combinations of deletions. However, studies of selected sets of genes, such as sets of paralogs with a greater a priori
chance of sharing a common function, would be informative.
In the yeast Saccharomyces cerevisiae
, gene knockout is accomplished by replacing a gene with a selectable marker via homologous recombination. Because the number of markers is limited, methods have been developed for removing and reusing the same marker5,6,7,8,9,10
. However, sequentially engineering multiple mutations using these methods is time-consuming because the time required scales linearly with the number of deletions to be generated.
Here we describe the Green Monster method for routinely engineering multiple deletions in yeast11
. In this method, a green fluorescent protein (GFP) reporter integrated into deletions is used to quantitatively label strains according to the number of deletions contained in each strain (Figure 1
). Repeated rounds of assortment of GFP-marked deletions via yeast mating and meiosis coupled with flow-cytometric enrichment of strains carrying more of these deletions lead to the accumulation of deletions in strains (Figure 2
). Performing multiple processes in parallel, with each process incorporating one or more deletions per round, reduces the time required for strain construction.
The first step is to prepare haploid single-mutants termed 'ProMonsters,' each of which carries a GFP reporter in a deleted locus and one of the 'toolkit' loci—either Green Monster GMToolkit-a
or GMToolkit-α at the can1Δ
locus (Figure 3
). Using strains from the yeast deletion collection12
, GFP-marked deletions can be conveniently generated by replacing the common KanMX4
cassette existing in these strains with a universal GFP
fragment. Each GMToolkit contains: either the a
- or α-mating-type-specific haploid selection marker1
and exactly one of the two markers that, when both GMToolkits are present, collectively allow for selection of diploids.
The second step is to carry out the sexual cycling through which deletion loci can be combined within a single cell by the random assortment and/or meiotic recombination that accompanies each cycle of mating and sporulation.
Microbiology, Issue 70, Genetics, Synthetic Biology, Environmental Genomics, Genomics, Bioengineering, Biomedical Engineering, Cellular Biology, Multi-site genomic engineering, genetic interaction, green fluorescent protein, GFP, flow cytometry, Saccharomyces cerevisiae, yeast, Green Monster
Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays
Institutions: University of Toronto, University of Toronto, University of Regina.
Phenotypes are determined by a complex series of physical (e.g.
protein-protein) and functional (e.g.
gene-gene or genetic) interactions (GI)1
. While physical interactions can indicate which bacterial proteins are associated as complexes, they do not necessarily reveal pathway-level functional relationships1. GI screens, in which the growth of double mutants bearing two deleted or inactivated genes is measured and compared to the corresponding single mutants, can illuminate epistatic dependencies between loci and hence provide a means to query and discover novel functional relationships2
. Large-scale GI maps have been reported for eukaryotic organisms like yeast3-7
, but GI information remains sparse for prokaryotes8
, which hinders the functional annotation of bacterial genomes. To this end, we and others have developed high-throughput quantitative bacterial GI screening methods9, 10
Here, we present the key steps required to perform quantitative E. coli
Synthetic Genetic Array (eSGA) screening procedure on a genome-scale9
, using natural bacterial conjugation and homologous recombination to systemically generate and measure the fitness of large numbers of double mutants in a colony array format.
Briefly, a robot is used to transfer, through conjugation, chloramphenicol (Cm) - marked mutant alleles from engineered Hfr (High frequency of recombination) 'donor strains' into an ordered array of kanamycin (Kan) - marked F- recipient strains. Typically, we use loss-of-function single mutants bearing non-essential gene deletions (e.g.
the 'Keio' collection11
) and essential gene hypomorphic mutations (i.e.
alleles conferring reduced protein expression, stability, or activity9, 12, 13
) to query the functional associations of non-essential and essential genes, respectively. After conjugation and ensuing genetic exchange mediated by homologous recombination, the resulting double mutants are selected on solid medium containing both antibiotics. After outgrowth, the plates are digitally imaged and colony sizes are quantitatively scored using an in-house automated image processing system14
. GIs are revealed when the growth rate of a double mutant is either significantly better or worse than expected9
. Aggravating (or negative) GIs often result between loss-of-function mutations in pairs of genes from compensatory pathways that impinge on the same essential process2
. Here, the loss of a single gene is buffered, such that either single mutant is viable. However, the loss of both pathways is deleterious and results in synthetic lethality or sickness (i.e.
slow growth). Conversely, alleviating (or positive) interactions can occur between genes in the same pathway or protein complex2
as the deletion of either gene alone is often sufficient to perturb the normal function of the pathway or complex such that additional perturbations do not reduce activity, and hence growth, further. Overall, systematically identifying and analyzing GI networks can provide unbiased, global maps of the functional relationships between large numbers of genes, from which pathway-level information missed by other approaches can be inferred9
Genetics, Issue 69, Molecular Biology, Medicine, Biochemistry, Microbiology, Aggravating, alleviating, conjugation, double mutant, Escherichia coli, genetic interaction, Gram-negative bacteria, homologous recombination, network, synthetic lethality or sickness, suppression
Aseptic Laboratory Techniques: Plating Methods
Institutions: University of California, Los Angeles .
Microorganisms are present on all inanimate surfaces creating ubiquitous sources of possible contamination in the laboratory. Experimental success relies on the ability of a scientist to sterilize work surfaces and equipment as well as prevent contact of sterile instruments and solutions with non-sterile surfaces. Here we present the steps for several plating methods routinely used in the laboratory to isolate, propagate, or enumerate microorganisms such as bacteria and phage. All five methods incorporate aseptic technique, or procedures that maintain the sterility of experimental materials. Procedures described include (1) streak-plating bacterial cultures to isolate single colonies, (2) pour-plating and (3) spread-plating to enumerate viable bacterial colonies, (4) soft agar overlays to isolate phage and enumerate plaques, and (5) replica-plating to transfer cells from one plate to another in an identical spatial pattern. These procedures can be performed at the laboratory bench, provided they involve non-pathogenic strains of microorganisms (Biosafety Level 1, BSL-1). If working with BSL-2 organisms, then these manipulations must take place in a biosafety cabinet. Consult the most current edition of the Biosafety in Microbiological and Biomedical Laboratories
(BMBL) as well as Material Safety Data Sheets
(MSDS) for Infectious Substances to determine the biohazard classification as well as the safety precautions and containment facilities required for the microorganism in question. Bacterial strains and phage stocks can be obtained from research investigators, companies, and collections maintained by particular organizations such as the American Type Culture Collection
(ATCC). It is recommended that non-pathogenic strains be used when learning the various plating methods. By following the procedures described in this protocol, students should be able to:
● Perform plating procedures without contaminating media.
● Isolate single bacterial colonies by the streak-plating method.
● Use pour-plating and spread-plating methods to determine the concentration of bacteria.
● Perform soft agar overlays when working with phage.
● Transfer bacterial cells from one plate to another using the replica-plating procedure.
● Given an experimental task, select the appropriate plating method.
Basic Protocols, Issue 63, Streak plates, pour plates, soft agar overlays, spread plates, replica plates, bacteria, colonies, phage, plaques, dilutions
Genetic Manipulation in Δku80 Strains for Functional Genomic Analysis of Toxoplasma gondii
Institutions: The Geisel School of Medicine at Dartmouth.
Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene function and phenotype(s). The development of mutant strains with targeted gene deletions, targeted mutations, complemented gene function, and/or tagged genes provides powerful strategies to address gene function, particularly if these genetic manipulations can be efficiently targeted to the gene locus of interest using integration mediated by double cross over homologous recombination.
Due to very high rates of nonhomologous recombination, functional genomic analysis of Toxoplasma gondii
has been previously limited by the absence of efficient methods for targeting gene deletions and gene replacements to specific genetic loci. Recently, we abolished the major pathway of nonhomologous recombination in type I and type II strains of T. gondii
by deleting the gene encoding the KU80 protein1,2
. The Δku80
strains behave normally during tachyzoite (acute) and bradyzoite (chronic) stages in vitro
and in vivo
and exhibit essentially a 100% frequency of homologous recombination. The Δku80
strains make functional genomic studies feasible on the single gene as well as on the genome scale1-4
Here, we report methods for using type I and type II Δku80Δhxgprt
strains to advance gene targeting approaches in T. gondii
. We outline efficient methods for generating gene deletions, gene replacements, and tagged genes by targeted insertion or deletion of the hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT
) selectable marker. The described gene targeting protocol can be used in a variety of ways in Δku80
strains to advance functional analysis of the parasite genome and to develop single strains that carry multiple targeted genetic manipulations. The application of this genetic method and subsequent phenotypic assays will reveal fundamental and unique aspects of the biology of T. gondii
and related significant human pathogens that cause malaria (Plasmodium
sp.) and cryptosporidiosis (Cryptosporidium
Infectious Diseases, Issue 77, Genetics, Microbiology, Infection, Medicine, Immunology, Molecular Biology, Cellular Biology, Biomedical Engineering, Bioengineering, Genomics, Parasitology, Pathology, Apicomplexa, Coccidia, Toxoplasma, Genetic Techniques, Gene Targeting, Eukaryota, Toxoplasma gondii, genetic manipulation, gene targeting, gene deletion, gene replacement, gene tagging, homologous recombination, DNA, sequencing
Combining Magnetic Sorting of Mother Cells and Fluctuation Tests to Analyze Genome Instability During Mitotic Cell Aging in Saccharomyces cerevisiae
Institutions: Rensselaer Polytechnic Institute.
has been an excellent model system for examining mechanisms and consequences of genome instability. Information gained from this yeast model is relevant to many organisms, including humans, since DNA repair and DNA damage response factors are well conserved across diverse species. However, S. cerevisiae
has not yet been used to fully address whether the rate of accumulating mutations changes with increasing replicative (mitotic) age due to technical constraints. For instance, measurements of yeast replicative lifespan through micromanipulation involve very small populations of cells, which prohibit detection of rare mutations. Genetic methods to enrich for mother cells in populations by inducing death of daughter cells have been developed, but population sizes are still limited by the frequency with which random mutations that compromise the selection systems occur. The current protocol takes advantage of magnetic sorting of surface-labeled yeast mother cells to obtain large enough populations of aging mother cells to quantify rare mutations through phenotypic selections. Mutation rates, measured through fluctuation tests, and mutation frequencies are first established for young cells and used to predict the frequency of mutations in mother cells of various replicative ages. Mutation frequencies are then determined for sorted mother cells, and the age of the mother cells is determined using flow cytometry by staining with a fluorescent reagent that detects bud scars formed on their cell surfaces during cell division. Comparison of predicted mutation frequencies based on the number of cell divisions to the frequencies experimentally observed for mother cells of a given replicative age can then identify whether there are age-related changes in the rate of accumulating mutations. Variations of this basic protocol provide the means to investigate the influence of alterations in specific gene functions or specific environmental conditions on mutation accumulation to address mechanisms underlying genome instability during replicative aging.
Microbiology, Issue 92, Aging, mutations, genome instability, Saccharomyces cerevisiae, fluctuation test, magnetic sorting, mother cell, replicative aging
Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast
Institutions: University of Uppsala, Swedish University of Agricultural Sciences.
Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using fluorochromes like Green Fluorescent Protein (GFP) directly attached to the protein of interest has become increasingly popular 1
. Using GFP and similar fluorochromes the subcellular localisations and movements of proteins can be detected in a fluorescent microscope. Moreover, also the subnuclear localisation of a certain region of a chromosome can be studied using this technique. GFP is fused to the Lac Repressor protein (LacR) and ectopically expressed in the cell where tandem repeats of the lacO
sequence has been inserted into the region of interest on the chromosome2
. The LacR-GFP will bind to the lacO
repeats and that area of the genome will be visible as a green dot in the fluorescence microscope. Yeast is especially suited for this type of manipulation since homologous recombination is very efficient and thereby enables targeted integration of the lacO
repeats and engineered fusion proteins with GFP 3
. Here we describe a quantitative method for live cell analysis of fission yeast. Additional protocols for live cell analysis of fission yeast can be found, for example on how to make a movie of the meiotic chromosomal behaviour 4
. In this particular experiment we focus on subnuclear organisation and how it is affected during gene induction. We have labelled a gene cluster, named Chr1, by the introduction of lacO
binding sites in the vicinity of the genes. The gene cluster is enriched for genes that are induced early during nitrogen starvation of fission yeast 5
. In the strain the nuclear membrane (NM) is labelled by the attachment of mCherry to the NM protein Cut11 giving rise to a red fluorescent signal. The Spindle Pole body (SPB) compound Sid4 is fused to Red Fluorescent Protein (Sid4-mRFP) 6
. In vegetatively growing yeast cells the centromeres are always attached to the SPB that is embedded in the NM 7
. The SPB is identified as a large round structure in the NM. By imaging before and 20 minutes after depletion of the nitrogen source we can determine the distance between the gene cluster (GFP) and the NM/SPB. The mean or median distances before and after nitrogen depletion are compared and we can thus quantify whether or not there is a shift in subcellular localisation of the gene cluster after nitrogen depletion.
Molecular Biology, Issue 59, Fission yeast, fluorescence microscopy, nuclear organisation, chromatin, GFP
High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay
Institutions: Massachusetts General Hospital.
We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson's disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla
luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla
luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.
Cellular Biology, Issue 88, Luciferases, Gene Transfer Techniques, Transfection, High-Throughput Screening Assays, Transfections, Robotics
A high-throughput method to globally study the organelle morphology in S. cerevisiae
Institutions: University of British Columbia - UBC.
High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help achieve an comprehensive understanding of molecular pathways. In Saccharomyces cerevisiae, with the development of the non-essential gene deletion array, we can globally study the morphology of different organelles like the endoplasmic reticulum (ER) and the mitochondria using GFP (or variant)-markers in different gene backgrounds. However, incorporating GFP markers in each single mutant individually is a labor-intensive process. Here, we describe a procedure that is routinely used in our laboratory. By using a robotic system to handle high-density yeast arrays and drug selection techniques, we can significantly shorten the time required and improve reproducibility. In brief, we cross a GFP-tagged mitochondrial marker (Apc1-GFP) to a high-density array of 4,672 nonessential gene deletion mutants by robotic replica pinning. Through diploid selection, sporulation, germination and dual marker selection, we recover both alleles. As a result, each haploid single mutant contains Apc1-GFP incorporated at its genomic locus. Now, we can study the morphology of mitochondria in all non-essential mutant background. Using this high-throughput approach, we can conveniently study and delineate the pathways and genes involved in the inheritance and the formation of organelles in a genome-wide setting.
Microbiology, Issue 25, High throughput, confocal microscopy, Acp1, mitochondria, endoplasmic reticulum, Saccharomyces cerevisiae


Developing a Wildfire Plan for Livestock
With the fires raging across the state, we are reminded once again of the risk wildfire poses to all of our farms and ranches. It is a good time to revisit (or create) a farm and ranch safety plan and update it with any missing information. If you have livestock, you will need to create an emergency plan for your animals.
There are five main components to your wildfire plan:
- Reducing fire risk
- Escape Routes
- Leaving Animals on the Property
- Other considerations
Reducing fire risk
One first step would be to set up a meeting with CalFire to review how to make your house, other structure, propane tank, and property more fire safe. You should consider creating an area of defensible space around your corrals/barns/small pastures adjacent to structures as well as your house. Disking at least ten feet around the perimeter would be a preferred option as well.
Determine if you have adequate water supply to fight the fire and wet down buildings. Is your source of water a well, irrigation ditch and /or pond? Do you have the ability to pump without power? This would mean a gas or diesel generator to ensure that you would be able to pump water. This would also mean adequate fuel to run the generator. You should still consider a backup generator even if you have gravity flow water because you may not have power for an extended period of time.
Do you have two separate ways in and out of the property? If not, is there a way to create an alternative route? If you only have one way in and out, what would you do if there was a fire approaching? You should contact friends or family that live out of your immediate area that may be able to temporarily house livestock. Fairgrounds and other facilities will also be available to house livestock during a wildfire evacuation. It is a great idea to contact local fairgrounds and facilities to learn more about temporary housing for livestock. Please note that you should contact friends, family, fairgrounds, and other potential facilities in the near future to discuss the situation.
The process is more than hauling animals and dropping them off. Where do you unload animals? Where will they be housed? What other feed, water troughs, and equipment do you need to bring? Is there someone who could temporarily feed and water while you were gone for a day or two trying to fight the fire on your property?
Leaving Animals on the Property
Many of us simply have too many animals and not enough transportation capacity to evacuate all of our animals from the path of an impending wildfire. Leaving animals out in a pasture, or “sheltering in place,” is a good option if the conditions allow animals to stay safe during a fire. According to the Humane Society of the United States, large animals may be able to shelter in place during a wildfire if a pasture area meets the following criteria:
- Over 1 acre in size,
- Has no overhead powerlines or poles,
- Has no barbed wire fencing (woven wire fencing is best).
- For high-wind scenarios, the pasture area must also be free of debris and easily uprooted trees.
You will also need to leave enough food and water for your animals to survive for a few days. You will not be able to return to your property to care for your animals until road closures and evacuation orders are lifted. Do not rely on automatic watering systems as power outages are likely in the event of a fire.
Many large animals are injured and killed attempting to escape from the path of a wildfire. Take down any portable fences and give your animals the largest space possible to move around in.
Do you have a designated backup in case you are away when the fire starts? This person would need familiarity with all of the above and live close by. Related to this is when to make the call to your backup person for hauling animals off the property. You must think quickly as conditions on a fire can change in less than hour. You should develop a decision-making process and a designated critical point that would mean implementing the fire plan.
Fires can happen suddenly and require rapid action. Keeping emotions in check will be critical to reacting effectively and staying calm. Normally calm animals will be excited by the fire and smoke and will be more difficult to handle. This will require patience to be sure they can be loaded in a trailer or herded to a safer location on the ranch as quickly as possible.
Situational awareness is a good concept to consider. This can be accomplished by developing a network of communication with other ranchers and landowners. Text messaging, email, or phone call can be an effective, quick way to inform others if you are seeing smoke or low flying airplanes as a first alert that a fire is in the area.
This is a good reference from New York that contains helpful checklists:
Additional information on wildfire evacuations are included here:


Former Danish colony, sold to the United States
They were seen by Columbus in 1493 and named after the Feast
of the Holy Virgin.
In 1555 they were claimed by Spain but actually settled by
the English and French with no real government, hence there was
piracy. All the natives had been killed by 1596 and replaced
by African slaves.
The western islands became Danish from about 1660. The United
States bought the islands in 1917. They are ruled by a governor
elected by the people and are a territory of the US.
Riots in 1989 following hurricane damage indicated considerable
discontent with the present situation and especially with the
incoming American settlers.


Patents in the Information Technology industry can be valuable as they protect innovative ideas and solutions to technical problems. By having a patent, a company can prevent others from using, selling, or manufacturing similar technology without their permission. This can provide a competitive advantage and help the company to establish itself as a market leader. Additionally, patents can also be licensed or sold for a profit, providing a source of revenue for the patent holder.
However, as always, we have to be careful: the value of a patent in the IT industry can also be limited by the speed at which technology is changing and the difficulties in enforcing patents in this field. As a result, the value of patents in IT can vary greatly and it is important to carefully consider the potential benefits and limitations before investing in them.
So, as you are aware, my team are working on multiple open source projects, morganstanley/ComposeUI: A .NET Core based, WebView2 using UI Container for hybrid web-desktop applications (github.com) is among them. Applying for a patent for an open source software may seem counterintuitive, as open source software is typically made available to the public under an open source license, allowing anyone to use, modify, and distribute the software. However, there may still be some reasons why a company or an individual might choose to apply for a patent for an open source software:
- Defense against patent trolls: Even though the software is open source, a company or individual may still apply for a patent to use as a defensive measure against patent trolls. Having a patent can help prevent others from making frivolous patent infringement claims.
- Commercializing the software: A company or individual may choose to apply for a patent as a way to commercialize the open source software. For example, the patent can be used to offer consulting services, support, or other value-added services related to the software.
- Protecting specific innovations: While the software is open source, there may be specific innovations within the software that the company or individual wants to protect. In this case, applying for a patent can help prevent others from using or commercializing these specific innovations.
So are there patented open source projects out there? Sure! An example of a patent for an open source software would be the “Method and System for Facilitating Electronic Transactions Over a Network” patent held by the OpenSSL Software Foundation. OpenSSL is an open source software that provides a secure way for websites to transmit data over the internet, and it is used by many websites and applications. Another example of a patent for an open source software is the “Method and System for Compression and Decompression of Data” patent held by the 7-Zip Software Foundation. 7-Zip is a free and open source file archiving software that is widely used for compressing and decompressing data. Despite being open source, the OpenSSL Software Foundation and the 7-Zip Software Foundation holds these patents to help protect the specific innovations in their software and to provide a defensive measure against patent trolls. By holding the patent, they can control how the technology is used and ensure that it remains available to the public under an open source license.
If you look from the other angel, there are other reasons why patenting open source software makes sense, like:
- To attract investment: By holding a patent, an open source software project can demonstrate its innovation and attract investment from potential partners or investors.
- To establish market position: Holding a patent can help establish a company or project as a market leader and give them a competitive advantage in their industry.
- To protect against infringement: Having a patent can provide legal protection against others who might use the technology without permission.
- To create a licensing revenue stream: An open source software project can license its patents to others, generating a new revenue stream that can be used to fund further development and improvement of the software.
It’s worth noting that these benefits of patenting open source software are not guaranteed, and each case is unique. The decision to patent open source software should be based on a careful consideration of the specific circumstances and goals of the company or individual.


Possibly the most underestimated principle of weight loss. YOU MUST DRINK AT LEAST 3 LITRES OF WATER EVERY DAY. Water is the most important compound in your body. Here are some of the functions performed by water in the body:
Top 5 reasons to drink water:
- It is the basis of all body fluids such as blood and saliva.
- Water keeps the skin hydrated, supple and looking healthy and glowing.
- Water is the main component of muscles and keeps them toned and firm.
- Water dilutes toxins and removes them from the body.
- Often we confuse needing water with being hungry. So drink water instead of snacking
There are steps you can take to drinking more water, carry a bottle of water with you every where you go. When out, always buy a drink and have a glass of water along side…
*Image courtesy of gt_pann/FreeDigitalPhotos.net


Computing at Jesse Gray
At Jesse Gray, we realise computers and technology are now part of everyday life. For most of us, this technology is essential to our lives, at home and at work. ‘Computational thinking’ is a skill that children must be taught if they are to be ready for the workplace and able to participate effectively in this digital world. At Jesse Gray, children learn how computers and computer systems work, they design and build programs, develop their ideas using technology and create a range of content. Computational thinking is an integral part of all computing lessons at Jesse Gray. It allows us to solve problems, design systems, and understand the power and limits of human and machine intelligence. It is a skill that empowers our children to develop their thinking. Our pupils are able to think computationally, they are therefore better able to conceptualise, understand and use computer-based technology, and so are better prepared for today’s world and the future.
Computing at Jesse Gray is a practical subject, in which invention and resourcefulness are encouraged. The ideas of computing are applied to understanding real-world systems and creating purposeful products. This combination of principles, practice and invention makes computing an extraordinarily useful and intensely creative subject, suffused with excitement, both visceral (‘it works!’) and intellectual (‘that is so beautiful’). Computing at Jesse Gray is not all screen based. Most lessons involve practical resources and technologies. Children use Sphero robots to create algorithms, controlling a physical robotic ball through their code. Through the use of iPads, children create stop motion movies, film and edit clips together using iMovie and create podcasts and commentaries explaining their learning.
At Jesse Gray, we support, develop and promote children’s Spiritual, Moral, Social and Cultural development through Computing. Computing develops children’s spiritual development by wondering at the power of the digital age through exploring how we use the internet, by understanding the advantages and limitations of technology and by using the internet as a gateway to big life issues. Computing develops children’s moral development by exploring the moral issues surrounding the use of data, by considering the benefits and potential dangers of the internet, and by understanding cyber bullying as a danger. Computing develops children’s social development by links through Twitter and other digital media services and by highlighting ways to stay safe when using online services and social media. Computing develops children’s cultural development by exploring human achievements and creativity in relation to worldwide communications and by developing a sense of awe and wonder at human ingenuity.
As a school that nurtures independent learners, we encourage our children to assess and understand their own ability in Computing. We provide children with a wide range of technologies, open-ended activities and the opportunities to work collaboratively on joint projects. As a dyslexic friendly school that values all learning styles, we provide our children with the opportunities to present the depth of their knowledge, skills and understanding through a variety of media. In computing, children use of a range of applications, such as Google Sites, PicCollage and iMovie as well as a variety of programs, such as Scratch, Robomind and MicroBits to showcase their learning. This means that when children’s English skills are not yet fully developed, children’s ability in Computing is not restricted.
To ensure the best Computing teaching and learning is consistent across the school, our Computing Leader and Technologies Team closely monitors planning, resources, lessons and assessment, providing CPD tailor made to the teaching and learning needs of Jesse Gray. Our staff are encouraged to share best practices and value pupil voice, ensuring that our children leave primary school with a love of Computing, and an integrated understanding of how technology can be used.


CONCEPTS/SKILLS: Shapes (Oval), Patterns & Art Media
- Students will create a spring egg out of pastels, construction paper and glue.
- Students will create and continue a pattern.
- Students will experience a different type of art media.
- Black Construction Paper (8 1/2 x 11)
- One Bottle of Elmer’s or Sparkle Glue per student
- One Box of Pastels (Colored Chalk) Per Table Group
- One Bottle of Hair Spray
- One Pencil Per Student
- Paint Shirts
Cut a traceable, egg shape pattern out of thick, cardstock. The egg pattern should be as tall as an 8 1/2 x 11 piece of construction paper. Trace at least one onto black construction paper per student. A few extras wouldn’t hurt!
Lesson Plan Outline:
- Have the student’s close their eyes and imagine an Easter egg. Ask: What colors do you see on the egg? What patterns/shapes do you imagine on the egg? What colors do you usually associate with Easter? Make a list on the board. (Explain what pastels are.)
- Pass out the black construction paper with the egg outlined on it. Ask: What do you think you are going to do with this?
- First have the kids write their names on the back. Next, have them whisper draw (draw lightly) patterns inside the egg outline. Circles, straight and squiggly lines are all OK. Make sure that they don’t draw the lines to close together or spend too much time on their patterns.
- Next, have the students cover their pencil lines with a bead of glue. Set aside and let dry over night. The glue will dry clear. The next day they will fill in-between the lines with pastel colors.
- Review pastels.
- Create a pattern using students in front of the class. Example, line up a boy, girl, boy, girl, etc. Ask the remaining students to identify the pattern.
- Show a spring egg and the pastel chalk. Ask: How could you create a pattern using these pastels and the spring egg?
- Demonstrate how to fill in-between the dry, glued, lines with pastels to create a pattern. (This is messy! Paint shirts should be worn.)
- Pass out their egg patterns and one box of pastels per table group.
- Lay the eggs in a well-ventilated area and spray with hair spray. The hair spray sets the pastels and prevents it from rubbing off when touched.
- Mount the spring eggs onto construction paper for display.
Grade Level(s): 1-2, 3-5
By: Terry Sayre


New trail marks Burgoyne's surrender
Victory Woods is lined with panels detailing the British Army's campaign
Published 1:00 am, Friday, June 4, 2010
VICTORY -- More than 230 years after Gen. John Burgoyne's surrender near present day Schuylerville, the event is being commemorated by the opening of Victory Woods, a trail next to the Saratoga Monument that details where Burgoyne spent his last days before being captured by colonial forces in 1777.
The 22-acres occupied by Victory Woods was donated to the Saratoga National Historical Park by the Victory Mills Packaging company in the 1970s. Nothing was done with it, however, until 1991 when Joe Finan, superintendent of the Saratoga national park, says he started pushing for funding for an archaeological assessment of the site. In 2004 he got funding for studies of the area and amenities for where the site is located.
Victory Woods is accessed by a tree-lined, old carriage path in the cemetery next to the Saratoga Monument. The site starts on a boardwalk that sits under a thick canopy of trees and the boardwalk leads through a marshy section of forest that Burgoyne and his men once occupied. The trail, a 1-mile loop, is lined with panels chronicling Burgoyne's Saratoga Campaign and the conditions leading to the British army's first surrender overseas.
Archaeological and landscape resources were researched for the site. Finan said the research team found artifacts from indigenous people who lived in the region over 6,000 years ago, but "really didn't find too many 18th-century artifacts at the site." He attributes this to archaeological looting. Because of the lack of military remains from the encampment, Finan said researchers were not able to determine exactly where different regiments from Burgoyne's army were located at the site.
Still, "the site stayed relatively undisturbed" according to Finan. By that, he means the remnants of the earthworks, or the army's fortifications made of wood and earth have not been disturbed since Burgoyne's surrender.
"We needed the earthworks displayed," Finan said. To do this, an ADA accessible boardwalk was built through Victory Woods. Clark Dalzell, ground and trail foreman, and his crew spent "the better part of three summers" working on the boardwalk, half of which is built on park property and the other on the town of Victory's property.
Building the trail presented problems with drainage and dealing with the mud of the marshy area, according to Dalzell. Finan said "it was a real challenge (carrying) all of the 4-by-4s in by hand."
After all of the effort, Dalzell says "the biggest reward is members of the community using the trail."
The trail will officially open at 11 a.m. Saturday with a ceremony featuring comments by public officials, followed by a talk by Park Historian Eric Schnitzer about the site's history, a ribbon cutting and a one mile walk around the trail. Soon after the trail's opening it will be linked to the Champlain Canal Trail and other developing trails in the area. Victory Woods will be open daily from sunrise to sunset and will feature occasional ranger guided hikes throughout the summer.


The Fatimid mosque in Saint Catherine’s Monastery, built in 500 AH/1106 AD during the reign of the Fatimid Caliph al-Amir bi-Ahkam Allah, was the fruit of the harmonious relationship between Muslims and Christians. The Fatimid Caliphate witnessed the peak of this relationship, and the caliphs of the period worked tirelessly to build mosques in sacred locations. The Fatimid mosque in Saint Catherine’s Monastery became a stop for pilgrims on their way to Mecca, many of whose writings can still be seen on its mihrab to this day.
The mosque lies in the north-western sector of Saint Catherine’s Monastery, opposite the Church of the Transfiguration of Christ the Savior, its principal church, such that its minaret stands side-by-side with the church’s steeple, in a perfect symbol of religious harmony. The mosque features several semi-circular vaults and three mihrabs. The mihrab is a semi-circular recess in the wall of a given mosque that indicates the qibla, the direction of the Kaaba in Mecca, which Muslims face during prayer. The principal mihrab is similar in its design to the one in the oldest section of al-Azhar mosque, and has a beautiful minbar, or pulpit, one of only three complete surviving examples from the Fatimid Period.


Every gardener knows that they ought to be making their own compost. Heck, even conscientious homeowners know they should be composting. The average American creates 1,600 pounds of waste every year and 65% of that is organic and could be composted rather than hauled off to a landfill.
Having plenty of rich compost on hand is also one of the very best things you can do for a garden. Plants that are somewhat sensitive like tomatoes absolutely love to start life in a big dollop of compost. Check out this guide to starting tomato plants for more tips on that front.
The thing that stopped me from composting for the longest time was just that it seems hard! Should I be using the hot method or the cold method? Do I have enough green versus brown material? Is it getting enough air? Enough water? What’s the carbon to nitrogen ratio?
This is all just stress you don’t need. Composting can be almost as easy as eating bon-bons while watching TV. Below I run through some of the world’s easiest methods for composting and give you a list of things you DON’T need to worry about.
Don’t Worry About Any of This
Below is a list of things that you see covered in many articles on the web, but with which you don’t really need to be bothered.
- Balancing ingredients – This is the whole green versus brown materials conundrum, but the thing to know is that balancing your materials is completely optional. This is important for commercial production but for your household needs, you can forget about it.
- Hot or Cold – This also doesn’t matter much in terms of whether you get compost, it’s just a matter of how fast you get it. Hot composting is faster but takes quite a bit more effort and expertise.
To Turn or Not to Turn – Turning compost gets oxygen through the material but as long as it’s not too wet, compost will aerate itself as it shrinks down.
- Size Doesn’t Matter – You hear a lot about how the compost pile needs to be 3 feet wide by 3 feet high, but that’s actually no guarantee that it will work better or faster. Your materials will rot regardless.
- Whether the Compost is Done – This one might be a surprise, but if you check your pile and the compost isn’t really done yet, no big deal. You can plant in immature compost and cover it with soil. You can also put a layer of it down in the fall and cover it over for use the following spring.
Now that I’ve put your mind at ease, here are a couple super simple methods to get your home composting practice off the ground.
The Lawnmower Composting Method
I think this is the single easiest method of composting that exists. If executed properly, you do virtually nothing.
1) Have the neighborhood kid who mows your lawn put all the lawn clippings into a tall pile and have him add to that pile every time the lawn is cut. (If you’re mowing your own lawn, you need to work on your laziness.)
2) Water that pile just enough to moisten it all the way through with your hose. This concludes the work on your part.
3) Wait 12-18 months and then use the compost for gardening.
Below is an excellent video showing this method in action. You’ll notice that despite the name of the video he does quite a lot of work with a pitchfork, all of which can be avoided if you don’t need your compost quickly.
The Trash Can Composting Method
Have one of those old metal trash cans? If not, you can still probably find one at the dump and you will have one of the cheapest and finest composters a lazy gardener can own.
1) Punch some holes in the bottom of the can with a hammer and a large nail.
2) Set the can on 4 bricks to keep it off the ground.
3) Thrown in a few shovels full of garden soil, some kitchen scraps and some leaves or grass clippings (about 3 inches of each).
4) Keep putting in more of the compost materials as you have them, keeping the lid on the can.
5) Within about 3 months you’ll have perfect compost, all with no watering or turning.
Composting is one of the most environmentally friendly practices you can incorporate into your household. It’s also one of the key factors that can massively increase the productivity of your garden. Hopefully you’ve seen just how easy this can be…even lazy gardeners can do it!


With the installation of a solar PV system, the facility now has electricity for the first time, and will be able to power lighting, fans, as well as electronic educational materials that expand the school's access to resources and enhance the overall learning environment for the students. This project is the second phase of GRID's effort to bring solar electricity to Juntas de Neji in 2018, and followed the installation of 18 solar home systems that were installed in November. Both phases of the project use Trojan batteries to store electricity generated by the solar panels.
For the upcoming 25 years, the community of Juntas de Neji and their future generations will finally have access to clean, reliable electricity for the first time. Access to electricity for the community and school of Juntas de Neji will dramatically improve the quality of life for residents, create an opportunity to generate income, and greatly enhance the learning environment and educational resources for the local children.
Check out photos this project here!


What is Twitter?
Twitter is used primarily as a method of communication made up of 140 characters called a ‘Tweet’. Tweets tend to reference people, places, and/or activities to which the said referee can respond. Tweeters either directly reference another person or broadcast information to which others can reply and respond.
Twitter users are able to follow or be followed. To follow somebody it ensures that all of their activity and comments appear in the followers news feed. The obvious benefit of having followers is that the information you broadcast is instantly distributed into their news feed. Users can also private message each other when they don’t want conversations to appear. @ClaypolePrimary will not enter into private discussions with others.
What is the primary purpose of @ClaypolePrimary?
@ClaypolePrimary will be used principally to be followed by staff, parents and other professionals in order to advertise the excellent work by staff, children, parents and governors. It may contain links to the schools other hosting sites, such as Claypole Church of England Primary School website
Similarly, it will also contain information detailing special events in school. The aim of this is to run alongside more traditional methods like sending home letters, and not to replace it.
Who controls content for @ClaypolePrimary?
The uploading of content for @ClaypolePrimary will be controlled by the Headteacher and Deputy Headteacher. They will be responsible for password protection and uploading of content. When this policy is next reviewed, it is hoped that more staff at Claypole Church of England Primary School will be trained on how to upload their own content to Twitter.
Who can follow @ClaypolePrimary?
At this moment in time, @ClaypolePrimary will not allow followers under the age of 21 and will limit its followers to teaching staff’s professional accounts, Governors, the community and parents of Claypole Church of England Primary School children. It will block followers found to be under the age of 21.
When @ClaypolePrimary receives a follow request it will make informed decisions on a case-by-case basis as to accept or decline. These decisions will be based upon a number of factors:
The name of the individual as read on their Twitter account and whether this name correlates to that of a parent on the SIMS database; is a member of the Claypole Church of England Primary School teaching staff; is a school governor; is a known and proper person;
The quality of the biography of each individual as read in the bio section of their Twitter account;
The images on view in the potential followers Twitter account.
In most cases, one or more of the above will be enough to accept a follow request. In the unlikely event that a parent or other suitable follower is inadvertently rejected, the Head teacher will hear each individual on a case-by-case basis. These hearings will usually involve the prospective follower making themselves known in person to the above named persons, with their Twitter details, and should take no more than a few minutes.
Who will @ClaypolePrimary follow?
In order to protect itself from inappropriate content being distributed into its news feed, @ClaypolePrimary will not actively seek to follow other users.
What is inappropriate content and referencing and how will it be dealt with?
@ClaypolePrimary welcomes any referencing, mentions, or interactions that posit the school in a positive light only. Therefore, Claypole Church of England Primary School deems any of the following as inappropriate:
Offensive language or remarks aimed at the school, its staff, parents, governors or others affiliated with the school;
Extremist views or radical views or opinions as in line with the prevent duty and new radicalisation policy
Unsuitable images or content posted into its feed;
Unsuitable images or content finding its way from another’s account into the @ClaypolePrimary feed.
Images or text that infringe upon copyright;
Comments that aim to undermine the school, its staff, parents, governors or others affiliated with the school.
Any inappropriate content will be deleted and its users will be removed, blocked, and, depending on the nature of the comment, reported to Twitter.
Furthermore, incidents of a more serious nature may be reported to the appropriate authority.
At the beginning of each academic year, each parent is asked for their permission to put their children’s image on the school website. These permissions now include Twitter (this is part of the home-school agreement). Where the parents do not wish their child’s image to appear on the school website or on Twitter, the school will aim to follow these wishes.


© KURT LINDSAY
Details that can help identify an owl:
Does the owl have ear tufts on the top of its head, or is it round-headed?
Is the owl relatively large, or close to the size of a duck? Or is it relatively small, closer to the size of a robin?
Eye and bill color
Are the eyes yellow or orange, or are they dark brown or black? Is the bill dark colored, or light colored?
What color are the owl's feathers? Does it have any other distinct markings, like spots?
Time of day
Owls are typically nocturnal or crepuscular. However, activity patterns can change seasonally and vary from one individual to another. Generally, more energy is required during the breeding season, which means owls are out hunting for longer periods.
In general, owls hunt in two ways:
Perching and pouncing — usually from a low perch, common among forest owls
Quartering — flying low over the ground, common among open-country owls
What is the habitat like where you saw the owl? Is it an open meadow? A wooded park? A thick forest? Near water? Urban or suburban, or a more rural or undeveloped area?
Look at a species distribution map in a bird field guide. You can also search online for what owl species are in that area.
Do you hear a high or low pitched 'hoot'? Is the owl calling with a rhythmic, slower-paced hooting, or a faster-paced single note? Or is the owl yelping, screaming, whistling, or barking?


Research Report 2013
The Murdoch Children’s Research Institute (MCRI) and Children’s Cancer Centre (CCC) at the Royal Children’s Hospital are extremely grateful for the support of CIKA in assisting our efforts to understand the mechanisms underlying childhood cancer. Our team of clinicians and researchers have several aims. Firstly, to discover the triggers (including environmental factors and genes) that lead to cancer development. Secondly, to understand why some children respond well to treatment and have a full cure, while others don’t. Finally, to discover why some children have a very toxic response to treatment.
There is now a general consensus that addressing these questions requires the detailed investigation of a large number of individual tumours. In the case of childhood tumours this means the need to study tissue samples removed from patients as part of their normal course of clinical investigation and treatment.
The CCC at the Royal Children’s Hospital is the largest centre for the treatment of paediatric cancers in Australia, which provides a unique opportunity to access rare solid and liquid tumours from paediatric patients. The CCC tissue bank is one of only three paediatric tumour tissue banks in Australia and is a collection of solid tumour tissue, bone marrow, blood and cerebral spinal fluid (CSF) preserved for use in future research. The samples, which can be stored indefinitely in ultra-cold freezers or in liquid nitrogen, are considered incredibly precious.
The objective of the CCC tissue bank is to assemble a collection of human biological materials linked to relevant health information for clinical investigation and ethically approved research. This involves working in collaboration with pathologists, oncologists, surgeons and other hospital personnel, to obtain patient consent, to collect samples and to gather associated clinical information about each participant and their corresponding specimens. The CCC tissue bank facilitates the supply of tissue samples to promote translation of biomedical research into advances in health care and personalised medicine.
The CCC tissue tank was established in 2006 and currently holds over two hundred solid tumour specimens in the form of tissue blocks and cryopreserved cell suspensions, the majority of which are held in frozen storage in a controlled and secure facility within the MCRI. Application is currently being made to access many thousands of archived formalin-fixed, paraffin-embedded tissue blocks stored in Anatomical Pathology at The Royal Children’s Hospital.


(Beyond Pesticides, March 13, 2017) Nearly 1 in 4 species of native bee is imperiled and at increasing risk of extinction. This, according to a new report from the Center for Biological Diversity (CBD), released earlier this month. The report is the first comprehensive review of the more than 4,000 native bee species in North America and Hawaii, and finds that more than half the species assessed are declining. With native bee decline increasing, advocates say it is imperative that action be taken to reduce toxic pesticide use and restore native habitats lost to chemical-intensive agriculture, urbanization, and climate change.
The new analysis, Pollinators in Peril: A systematic status review of North American and Hawaiian native bees, reveals that more than 700 species are in trouble from a range of serious threats, including severe habitat loss and escalating pesticide use. Key findings include: (1) among native bee species with sufficient data to assess (1,437), more than half (749) are declining; (2) nearly 1 in 4 (347 native bee species) is imperiled and at increasing risk of extinction; (3) many of the bee species lacking sufficient data are also likely declining or at risk of extinction, highlighting the urgent need for additional research; and, (4) the declines are caused primarily by habitat loss, heavy pesticide use, climate change and urbanization.
These findings come as a growing body of research has revealed that more than 40 percent of insect pollinators are highly threatened globally, including many of the native bees critical to crop and wildflower pollination across the U.S. Many studies link pesticide use to these declines. Pesticides, like the neonicotinoid insecticides, have been shown to impair bee foraging and learning behavior, reproduction, and suppress bee immune systems making them more susceptible to disease and parasites. See ‘What the Science Shows.’
“The evidence is overwhelming that hundreds of the native bees we depend on for ecosystem stability, as well as pollination services worth billions of dollars, are spiraling toward extinction,” said Kelsey Kopec, a native pollinator researcher and author of the study. “It’s a quiet but staggering crisis unfolding right under our noses that illuminates the unacceptably high cost of our careless addiction to pesticides and monoculture farming.”
Honey bee decline has been much discussed in recent years. Last winter (2015/2016), beekeepers lost 44 percent of their honey bee colonies. But, until now, much less has been revealed about the 4,337 native bee species in North America and Hawaii. These mostly solitary, ground-nesting bees play a crucial ecological role by pollinating wild plants, and provide more than $3 billion in fruit-pollination services each year in the U.S. To assess current populationstopped trends and threats as comprehensibly as possible for the 4,337 described species of North American and Hawaiian bees, the CBD report reviewed the current conservation status of 316 species as established by state, federal, or independent research.
The report highlights five imperiled native bees that offer a snapshot of the threats driving declines in many native bee species:
- Yellow carpet solitary bee: This dark, olive-green bee, whose fate is intertwined with its floral host and California’s dwindling vernal pools, is severely threatened with extinction.
- Sunflower leafcutting bee: This spectacularly large bee used to be seen patrolling sunflower stands throughout the Great Plains; it is now in steep decline and rarely seen.
- Wild sweet potato bee: Known for its unique three-lobed snout, this bee, once commonly seen foraging across much of the East, is now dangerously imperiled.
- Gulf Coast solitary bee: Completely dependent on the disappearing coastal plain honeycombhead plant and the barrier-island sand dunes where it nests, this bee is now found only within a shrinking portion of its range along the Gulf Coast.
- Macropis cuckoo bee: This nest invader, which takes over the nests of other bee species to lay its eggs, was once common across much of central and eastern North America but is now considered that region’s most endangered bee.
Earlier this year, the Rusty Patched Bumblebee became the first bumblebee federally designated as endangered under the Endangered Species Act. The Rusty Patched Bumblebee was once widespread throughout the U.S. and parts of Canada, but declined dramatically over the last couple decades. Now their populations are estimated to be less than 10% of what they once were. But, the Trump administration in February reversed the final decision, pending further review, for this listing. In response, the Natural Resources Defense Council (NRDC) filed a lawsuit charging the administration violated the notice and comment requirements of public rulemaking for the delay on the bumblebee listing.
With the decline of both native and managed bees, Beyond Pesticides is working to promote the widespread transition of conventional farmland to organic production. Organic law requires farmers to foster soil health, and create a strategy to prevent pest populations before they become a problem. Because of these factors, many certified organic farms do not need to use toxic pesticides because their required organic systems plan practices increase soil and plant health and pest and disease resiliency through an increased diversity of pest predators.
With one in three bites of food reliant on bees, other insects, and birds for pollination, the decline in pollinators due to pesticides, and other human-made causes, demands immediate action. For more on this and what you can do to protect pollinators, visit Beyond Pesticides’ BEE Protective webpage.
All unattributed positions and opinions in this piece are those of Beyond Pesticides.
Source: CBD press release and report


By Alyssa Keimach
Earth’s neighborhood just got a little larger.
Astronomers thought that Earth was located on a spur of an arm of the Milky Way… until data from the Very Long Baseline Array telescopes suggested that we could be located closer to the center.
While it’s simple to observe a bird’s-eye view of other galaxies, models of the Milky Way are inaccurate due to Earth’s limited vantage point. We are attempting to measure an entire galaxy using only Earth’s narrow perspective, and at the center of our galaxy is a large bulge, blocking about half of the Milky Way from view.
To make the best model possible, astronomers use a technique called parallax. Measurements are taken from locations on either side of the sun to give multiple perspectives of our location in the sky. Then, astronomers use trigonometry to calculate where we might reside in comparison to distant objects.
At the center of the Milky Way is a supermassive black hole, whose gravitational pull is capable of keeping 200–400 billion stars in orbit around the galaxy. Measurements from NASA’s Spitzer Space Telescope revealed that these stars are oriented in two arms that spiral around the black hole.
"Based on both the distances and the space motions we measured, our Local Arm is not a spur,” said Alberto Sanna, a postdoctoral fellow with the Max-Planck Institute for Radio Astronomy (MPIFR). “It is a major structure, maybe a branch of the Perseus Arm, or possibly an independent arm segment."
Sanna and his colleagues presented their research this week at the American Astronomical Society meeting, held in Indiana.
Astronomers are creating increasingly accurate models of the Milky Way and every new finding tells us more about the entire universe.
Alyssa Keimach is an astronomy and astrophysics student at the University of Michigan and interns for the Morrison Planetarium.


Dr. Larkin's research focuses on the molecular and chemical ecology of native plant species, in particular their interaction with their environment and response to habitat fragmentation. It includes both specific events, such as the effect of temperature on the expression of allelic variants of a single enzyme (Granule Bound Starch Synthase) and broader events, such as the genetic relationships among populations in fragmented habitats. Some of the plant species studied include rice (Oryza sativa L.), shoalgrass (Halodule beaudettei), turtlegrass (Thalassia testudinum) bundleflower (Desmanthus virgatus) and Kleberg's bluestem (Dichanthium annulatum). His research involves characterization of genomic and organellar DNA variation and expression through DNA sequencing, Amplified restriction Fragment Length Polymorphism (AFLP), Random Amplification of Polymorphic DNA (RAPD), and Microsatellite (STR) analysis. Field, molecular and environmental data are used to examine how plants respond and, eventually, adapt to their environments.