Abstract
Genetically modified mouse strains derived from embryonic stem (ES) cells have become essential tools for functional genomics and biomedical research. Large scale mutagenesis projects are producing libraries of mutant C57BL/6 (B6) ES cells to enable the functional annotation of every gene of the mouse genome. To realize the utility of these resources, efficient and accessible methods of generating mutant mice from these ES cells are necessary. Here, we describe a combination of ICR morula aggregation and a chemically-defined culture medium with widely available and accessible components for the high efficiency generation of germline transmitting chimeras from C57BL/6N ES cells. Together these methods will ease the access of the broader biomedical research community to the publicly available B6 ES cell resources.
Highlights
The generation of genetically modified mouse strains by homologous recombination in embryonic stem (ES) cells is a major research tool in diverse fields of biology
The four culture media tested were standard FBS-DMEM, where the FBS had been lot selected based on the ability to support completely ES cell-derived embryo and pup formation with F1 hybrid-derived (G4) ES cells in the tetraploid complementation assay; RESGROTM medium (Millipore); VGB6 conditioned medium, a proprietary formulation generously provided by Regeneron Pharmaceuticals, Inc. (Tarrytown, NY); and a defined medium comprised of KnockOutTM DMEM (Invitrogen) with KnockOutTM Serum Replacement (Invitrogen) and supplemented with two inhibitors (2i) [20,21] (KOSR+2i; see Materials and Methods)
The culture media were tested by passaging parental C2 ES cells in the media and testing their ability to generate chimeric animals based on coat colour contribution
Summary
The generation of genetically modified mouse strains by homologous recombination in embryonic stem (ES) cells is a major research tool in diverse fields of biology. Germline transmission breeding and subsequent backcrosses to C57BL/6 (B6) to generate congenic strains delay functional studies and increase costs, while at the same time genes closely linked to the original targeted locus remain from the original ES cell genome and may confound research results [4]. Together, these considerations led the large scale mouse mutagenesis projects organized under the umbrella of the International Knockout Mouse Consortium (IKMC) to select B6-derived ES cells as the parental lines in which to mutate all protein coding genes of mouse [5,6]. Microarray analysis of gene expression patterns in cultured 129- and B6-derived ES cells showed that B6 ES cells have a greater tendency to lose their pluripotency in culture in standard 15% FBS LIF-supplemented ES cell medium than 129 ES cells [16]
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