Germ-line transmission of a c-abl mutation produced by targeted gene disruption in ES cells. Sci NY NY

Department of Biochemistry and Molecular Biophysics, Columbia University, College of Physicians & Surgeons, New York, NY 10032.
Science (Impact Factor: 33.61). 12/1989; 246(4931):799-803. DOI: 10.1126/science.2554496
Source: PubMed

ABSTRACT A substitution mutation has been introduced into the c-abl locus of murine embryonic stem cells by homologous recombination between exogenously added DNA and the endogenous gene, and these cells have been used to generate chimeric mice. It is shown that the c-abl mutation was transmitted to progeny by several male chimeras. This work demonstrates the feasibility of germ-line transmission of a mutation introduced into a nonselectable autosomal gene by homologous recombination.

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Available from: Pamela L Schwartzberg, Jul 04, 2014
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    • "The first mouse alleles that were modified through targeted homologous recombination (HR) in ES cells were designed to inactivate gene function completely (Doetschman et al., 1988; Mansour et al., 1988; Thomas and Capecchi, 1987). The earliest of these targeted mutations involved extensive alterations to gene structure , most often produced through the deletion of coding exons and the insertion of drug selectable marker cassettes into target sites (DeChiara et al., 1990; Koller and Smithies, 1989; Schwartzberg et al., 1989; Zijlstra et al., 1989). Today, the constitutive or conditional inactivation of gene function remains a primary objective for the majority of engineered mouse alleles, and the efforts of individual labs have collectively resulted in the production of knockout alleles for several thousand genes (Eppig et al., 2012). "
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    ABSTRACT: Homologous recombination in embryonic stem (ES) cells offers an exquisitely precise mechanism to introduce targeted modifications to the mouse genome. This ability to produce specific alterations to the mouse genome has become an essential tool for the analysis of gene function and the development of mouse models of human disease. Of the many thousands of mouse alleles that have been generated by gene targeting, the majority are designed to completely ablate gene function, to create conditional alleles that are inactivated in the presence of Cre recombinase, or to produce reporter alleles that label specific tissues or cell populations (Eppig et al., 2012). However, there are a variety of powerful motivations for the introduction of subtle, targeted mutations (STMs) such as point mutations, small deletions, or small insertions into the mouse genome. The introduction of STMs allows the ablation of specific transcript isoforms, permits the functional investigation of particular domains or amino acids within a protein, provides the ability to study the role of specific sites with in cis-regulatory elements, and can result in better mouse models of human genetic disorders. In this review, I examine the current strategies that are commonly used to introduce STMs into the mouse genome and highlight new gene targeting technologies, including TALENs and CRISPR/Cas, which are likely to influence the future of gene targeting in mice. © 2013 Wiley Periodicals, Inc.
    genesis 08/2013; 51(9). DOI:10.1002/dvg.22422 · 2.02 Impact Factor
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    • "This was in accordance with the data on chimera production. Reports showed that the combination of the genotypes of ES cells and host blastocysts is important for production of germline transmission chimeras, and the C57BL/6 are the suitable host for 129-derived ES cells [3,34]. In this study, ES cells were derived from 129 mice and had been cultured in vitro for a long time (over 35 passages). "
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    ABSTRACT: Background Production of chimeric mice is a useful tool for the elucidation of gene function. After successful isolation of embryonic stem (ES) cell lines, there are many methods for producing chimeras, including co-culture with the embryos, microinjection of the ES cells into pre-implantation embryos, and use of tetraploid embryos to generate the full ES-derived transgenic mice. Here, we aimed to generate the transgenic ES cell line, compare the production efficiency of chimeric mice and its proportion to yield the male chimeric mice by microinjected ES cells into 4- to 8-cell and blastocysts embryos with the application of Piezo-Micromanipulator (PMM), and trace the fate of the injected ES cells. Results We successfully generated a transgenic ES cell line and proved that this cell line still maintained pluripotency. Although we achieved a satisfactory chimeric mice rate, there was no significant difference in the production of chimeric mice using the two different methods, but the proportion of the male chimeric mice in the 4- to 8-cell group was higher than in the blastocyst group. We also found that there was no tendency for ES cells to aggregate into the inner cell mass using in vitro culture of the chimeric embryos, indicating that they aggregated randomly. Conclusions These results showed that the PMM method is a convenient way to generate chimeric mice and microinjection of ES cells into 4- to 8-cell embryos can increase the chance of yielding male chimeras compared to the blastocyst injection. These results provide useful data in transgenic research mediated by ES cells.
    Journal of Animal Science and Biotechnology 03/2013; 4(1):12. DOI:10.1186/2049-1891-4-12 · 1.68 Impact Factor
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    • "There are several factors that have been demonstrated to affect the ES cell’s ability to transmit their genetic material through the germline including the genetic background, stemness, normality of karyotype, pathogen status of the ES cell line as well as the genetic background of recipient embryos [7], [21]. Among these factors, the combination of the genetic background of the ES cells and the recipient embryos has been demonstrated to be a critical factor affecting the germline transmissibility of the ES cells [7], [21], [22]. Ideally, the host background should allow the ES cells to have an optimal developmental advantage when injected into the blastocyst. "
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    ABSTRACT: Embryonic stem (ES) cell-based gene manipulation is an effective method for the generation of mutant animal models in mice and rats. Availability of germline-competent ES cell lines from inbred rat strains would allow for creation of new genetically modified models in the desired genetic background. Fischer344 (F344) males carrying an enhanced green fluorescence protein (EGFP) transgene were used as the founder animals for the derivation of ES cell lines. After establishment of ES cell lines, rigorous quality control testing that included assessment of pluripotency factor expression, karyotype analysis, and pathogen/sterility testing was conducted in selected ES cell lines. One male ES cell line, F344-Tg.EC4011, was further evaluated for germline competence by injection into Dark Agouti (DA) X Sprague Dawley (SD) blastocysts. Resulting chimeric animals were bred with wild-type SD mates and germline transmissibility of the ES cell line was confirmed by identification of pups carrying the ES cell line-derived EGFP transgene. This is the first report of a germline competent F344 ES cell line. The availability of a new germline competent ES cell line with a stable fluorescence reporter from an inbred transgenic rat strain provides an important new resource for genetic manipulations to create new rat models.
    PLoS ONE 02/2013; 8(2):e56518. DOI:10.1371/journal.pone.0056518 · 3.23 Impact Factor
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