As indicated earlier in this chapter, one side product of many transgenic experiments is the generation of mice in which a transgene insertion has disrupted an endogenous gene with a consequent effect on phenotype. Unlike spontaneous or mutagen-induced mutations, "insertional mutations" of this type are directly amenable to molecular analysis because the disrupted locus is tagged with the transgene construct. Unexpected insertional mutations have provided instant molecular handles not only for interesting new loci but for classical loci, as well, that had not been cloned previously (Meisler, 1992).
When insertional mutagenesis, rather than the analysis of a particular transgene construct, is the goal of an experiment, one can use alternative experimental protocols that are geared directly toward gene disruption. The main strategies currently in use are based on the introduction into ES cells of beta-galactosidase reporter constructs that either lack a promoter (Gossler et al., 1989; Friedrich and Soriano, 1991) or are disrupted by an intron (Kramer and Erickson, 1981). The constructs can be introduced by DNA transfection or within the context of a retrovirus (Robertson, 1991). It is only when a construct integrates into a gene undergoing transcriptional activity that functional beta-galactosidase is produced, and producing cells can be easily recognized by a color assay. Of course, the production of "beta-gal" will usually mean that the normal product of the disrupted gene can not be made and thus this protocol provides a means for the direct isolation of ES cells with tagged mutations in genes that function in embryonic cells. Mutant cells can be incorporated into chimeric embryos for the ultimate production of homozygous mutant animals that will display the phenotype caused by the absence of the disrupted locus. This entire technology, referred to as "gene trapping" (Joyner et al., 1992), is clearly superior to traditional chemical methods for the production of mutations at novel loci.
A computerized database (called TBASE) has been developed to help investigators catalog the strains that they produce and find potentially useful strains produced by others (Woychik et al., 1993). The database is available over the Internet through the Johns Hopkins Computational Biology Gopher Server and is linked to the on-line mouse databases maintained by the Jackson Laboratory Informatics Group (see Appendix B).
Although the gene replacement technology has been employed with success by increasing numbers of laboratories, it is still the case, and likely to remain so, that an enormous amount of time and effort goes into the production of each newly engineered mouse strain. Clearly, it does not make sense to derive strains with the same gene replacement more than once. However, with the high costs of animal care and maintenance, it is often difficult for researchers to maintain strains that they are no longer actively using. Furthermore, many individual research colonies are contaminated by various viruses, and as such, virus-free facilities are reluctant to import mice from anywhere other than reputable dealers. The Jackson Laboratory has recently come to the rescue by setting up a clearing house to preserve what are likely to be the most useful of these strains for other members of the worldwide research community. For the first time, JAX will be importing mice from large numbers of individual researchers. Each strain will be rederived by Caesarean section into a germ-free barrier facility, and will be made available for a nominal cost, without experimental restriction, to all members of the research community.
With the various technologies that have now been developed to manipulate the genomes of embryonic cells combined with ever more sophisticated molecular tools, it can be stated without exaggeration that the sky is the limit for what can be accomplished with the mouse as a model genetic system. It is always impossible to predict what the future holds, but one can imagine the use of both gene addition and gene replacement technologies as routine tools for assessing the functions of sequential segments of DNA obtained by walking along each mouse and human chromosome. With recent reports of success in the insertion of intact yeast artificial chromosome (YAC)-sized DNA molecules of 250 kb or more in length into the germline of transgenic animals, it becomes feasible to analyze even larger chunks of DNA for the presence of interesting genetic elements (Jakobovits et al., 1993; Schedl et al., 1993). In fact, it is only with experiments of this type that it will be possible to completely uncover all of the pathways through which a gene is regulated, and all of the pathways through which a gene product may function. Just as the study of neurons in isolation can not possibly provide a clue to human consciousness, the study of individual genes outside of the whole animal can not possibly provide a clue to the network of interactions required for the growth and development of a whole mouse or person.