Introduction

The development of murine embryonic stem (ES) cells has brought about another revolution in the biological sciences. When considering the most important experiments of the last century, one must include those resulting in the discovery that clones derived from the inner cell mass of early embryos could be propagated in culture and that resultant cells could contribute to the germ line after reinjection into blastocysts (Evans and Kaufman, 1981; Bradley et al., 1984; Robertson et al., 1986). The marriage of pluripotent ES cells with an understanding of gene targeting through homologous recombination was the additional breakthrough that allowed application of this powerful approach to the murine system (Doetschman et al., 1988; Mansour et al., 1988).

As a result of these advances, a number of truly remarkable experimental avenues are now available to the general scientific population. In essence, ES cells and related gene targeting technologies allow us to alter any locus within the mouse genome, in essentially any way that is desired. The results of this new-found ability include the generation of better mouse models of human disease and the capacity to examine mutations within a whole-animal context. Moreover, the cell lines and organ culture models that can be derived from these experiments provide powerful systems for the analysis of gene-function relationships in simpler in vitro systems.

Like any new methodology, ES cells and gene targeting can be technically demanding. Because of this challenge, one can make the mistake of being intimidated and never attempting such experiments for oneself. In the early years of these techniques, avoidance of ES cell biology and gene targeting was understandable. ES cell lines were not readily available, generation of mouse embryonic fibroblasts required a large up-front commitment, serum lots had to be checked carefully for their capacity to maintain ES cell pluripotency, and the procurement of targeting vectors and related reagents was difficult. Add to this the fact that only a few experts knew what an ES cell colony looked like further sowed the seeds of intimidation. Today, avoidance of this technology is not as defensible. Pluripotent ES cell lines, the required feeder cells, ES-validated serum lots, and parent targeting vectors are available from a number of commercial vendors. In addition, most universities have core facilities that can easily handle the injection needs of the average investigator. With these technical obstacles out of the way, the only impediment left is the normal energy barrier that prevents us from beginning any new technique in the lab.

It is certain that ES cell technology will soon become a standard approach in the field of toxicology. At the very least, toxicologists will be able to benefit greatly from the use of mutant mice that have been generated by others. The utility of this technology to toxicology is less of a prediction than an observation. Already, null alleles at Cyp loci have proven that certain monooxygenases play a role in the toxicity of various chemicals (e.g., Lee et al., 1996; Zaher et al., 1998; Buters et al., 1999). Similarly, null alleles at loci encoding various cytokines are demonstrating a role for these factors in the mechanisms of certain toxicants (e.g., Ladenheim et al., 2000).

The protocols that follow should provide a solid start to those individuals ready to establish ES cell technology in their own laboratories. Although these protocols are fairly detailed in nature, where appropriate, we have also tried to cite additional references and manuals in ES cell and mouse biology. In addition, a considerable discussion of ES cell biology can be found in unit 1.3. Here, we start with a section describing the general maintenance of ES cells, as well as the preparation of necessary feeder cells and related reagents (unit 15.1. This is followed with a section describing protocols for the rapid genotyping of ES cells after a gene-targeting event (unit 15.2). The current version of this chapter also includes a description of how an investigator can make aggregation chimeras (unit 15.3), this protocol allows the study of chimeric mice and serves as a starting point for germ-line transmission. Additionally, we have begun to address how an investigator can examine Cre-expression methods in recombinant mice through the use of novel and widely available “reporter mice” (unit 15.4). In future supplements, we will address the construction of targeting vectors and the fundamentals of ES cell microinjection into blastocysts.