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  • Colony Picking

    Document Type:
    Protocols
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  • High-throughput trapping of secretory pathway genes in mouse embryonic stem cells. 16478711

    High-throughput gene trapping is a random approach for inducing insertional mutations across the mouse genome. This approach uses gene trap vectors that simultaneously inactivate and report the expression of the trapped gene at the insertion site, and provide a DNA tag for the rapid identification of the disrupted gene. Gene trapping has been used by both public and private institutions to produce libraries of embryonic stem (ES) cells harboring mutations in single genes. Presently, approximately 66% of the protein coding genes in the mouse genome have been disrupted by gene trap insertions. Among these, however, genes encoding signal peptides or transmembrane domains (secretory genes) are underrepresented because they are not susceptible to conventional trapping methods. Here, we describe a high-throughput gene trapping strategy that effectively targets secretory genes. We used this strategy to assemble a library of ES cells harboring mutations in 716 unique secretory genes, of which 61% were not trapped by conventional trapping, indicating that the two strategies are complementary. The trapped ES cell lines, which can be ordered from the International Gene Trap Consortium (http://www.genetrap.org), are freely available to the scientific community.
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    Reference
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  • Optimization of Protocols for Derivation of Mouse Embryonic Stem Cell Lines from Refractory Strains, Including the Non Obese Diabetic Mouse. 21933027

    The derivation of pluripotent embryonic stem cells (ESCs) from a variety of genetic backgrounds remains a desirable objective in the generation of mice functionally deficient in genes of interest and the modeling of human disease. Nevertheless, disparity in the ease with which different strains of mice yield ESC lines has long been acknowledged. Indeed, the generation of bona fide ESCs from the non obese diabetic (NOD) mouse, a well-characterized model of human type I diabetes, has historically proved especially difficult to achieve. Here, we report the development of protocols for the derivation of novel ESC lines from C57Bl/6 mice based on the combined use of high concentrations of leukemia inhibitory factor and serum-replacement, which is equally applicable to fresh and cryo-preserved embryos. Further, we demonstrate the success of this approach using Balb/K and CBA/Ca mice, widely considered to be refractory strains. CBA/Ca ESCs contributed to the somatic germ layers of chimeras and displayed a very high competence at germline transmission. Importantly, we were able to use the same protocol for the derivation of ESC lines from nonpermissive NOD mice. These ESCs displayed a normal karyotype that was robustly stable during long-term culture, were capable of forming teratomas in vivo and germline competent chimeras after injection into recipient blastocysts. Further, these novel ESC lines efficiently formed embryoid bodies in vitro and could be directed in their differentiation along the dendritic cell lineage, thus illustrating their potential application to the generation of cell types of relevance to the pathogenesis of type I diabetes.
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    Reference
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  • Karyotyping ES Cells

    Document Type:
    Protocols
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  • Short RNAs are transcribed from repressed polycomb target genes and interact with polycomb repressive complex-2. 20542000

    Polycomb proteins maintain cell identity by repressing the expression of developmental regulators specific for other cell types. Polycomb repressive complex-2 (PRC2) catalyzes trimethylation of histone H3 lysine-27 (H3K27me3). Although repressed, PRC2 targets are generally associated with the transcriptional initiation marker H3K4me3, but the significance of this remains unclear. Here, we identify a class of short RNAs, approximately 50-200 nucleotides in length, transcribed from the 5' end of polycomb target genes in primary T cells and embryonic stem cells. Short RNA transcription is associated with RNA polymerase II and H3K4me3, occurs in the absence of mRNA transcription, and is independent of polycomb activity. Short RNAs form stem-loop structures resembling PRC2 binding sites in Xist, interact with PRC2 through SUZ12, cause gene repression in cis, and are depleted from polycomb target genes activated during cell differentiation. We propose that short RNAs play a role in the association of PRC2 with its target genes.
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    Reference
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  • Using small molecules to improve generation of induced pluripotent stem cells from somatic cells 20336525

    Induction of pluripotent stem cells from somatic cells by defined factors was shown to be possible only recently, but already several laboratories have made tremendous strive toward improving and understanding the process. Originally, Oct4, Sox2, Klf4, and cMyc were identified as being the combination of genes necessary to induce reprogramming. It was later shown that cMyc was dispensable; however, in its absence the process was less efficient and took a considerably longer period of time to occur. Furthermore, others have shown that the combination of Oct4, Sox2, Nanog, and Lin28 could also induce reprogramming. One major caveat associated with these techniques remains the need for overexpression of several genes using viral systems. Until very recently, most studies were done using integrating viruses such as retroviruses and lentiviruses. This method ensured that the protein of interested would be expressed at a high concentration and for an adequate period of time necessary to induce reprogramming. Up to date, others have now been able to use different nonintegrative method such as adenovirus and plasmid transfection to induce reprogramming. Furthermore, piggyBac transposition was successfully used to induce reprogramming of murine cells. Most importantly, it was recently published that reprogramming can be induced in the absence of virus, with proteins and small molecules. All of the later methods are appealing since they do not require the integration of the virus or plasmid to exert its effect. However, one avenue that would be all the more therapeutically appealing would be to induce reprogramming in the absence of gene overexpression systems, using small molecules to modulate signaling pathways in the somatic cells. A few molecules have already been identified with the ability to either improve the process or replace one or two of the genes deemed necessary for reprogramming. We have screened successfully for compounds that can replace some of these factors, and share the methods developed following these screens.
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    Reference
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