Abstract

Abstract The reintroduction of mouse ES (mES) cells into an embryonic environment allows expression of their pluripotency, as defined by the capacity to contribute to all tissue and cell types, including the germ cells (1, 2). This most powerful feature of mES cells has led to their exploitation in transgenic research, providing a direct route for the insertion of genetic modifications into the germline of fertile animals. Techniques for generating chimeric mice using mES cells – specifically, blastocyst injection and morula aggregation – are now standard worldwide (3); and recent refinements to those techniques have increased the efficiency of production of mice that are derived entirely – rather than partially – from the introduced mES cells, that is ‘mES cell-derived mice’. These refinements include the use of host embryos that have been rendered tetraploid (4), and which in consequence are incompetent to undergo fetal development; and the hyperthermic treatment of host embryos selectively to kill their inner cell masses (ICMs) (5). To compromise the viability of the host embryo’s ICM by either of these means favours its reconstitution by exogenously added mES cells, and consequently the production of mES cell-derived fetuses and offspring. In addition, mES cell-derived mice may be cloned by the technique of nuclear transfer using mES cells as the donor cells, or ‘karyoplasts’ (6). In this chapter we provide our current methods for producing mES cell-derived mice by embryo reconstitution (morula aggregation and blastocyst injection) using tetraploid and hyperthermically treated host embryos, and by nuclear transfer. Owing to space limitations, the reader is referred to primary texts for comprehensive coverage of supplementary methods in mouse husbandry, embryo micromanipulation, and surgical transfer (7).

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