Aging Aging

364 Andersenwhich resulting animals are chimeras, that is, made up of tissues from both ESand host cells. Following implantation into foster mothers, the resulting chimericoffspring are bred, producing mice that, if the ES cells have become partof the germ line, are heterozygous for the introduced mutation. Heterozygousanimals are bred to obtain homozygous mutant mice.5. Generation of a Particular Genetic Mutation via Gene TargetingSometimes, a specific subtle mutation in the endogenous gene is desiredrather than generation of a neoR insertional mutation. This requires a two-stepdouble gene replacement. First the mutant is made containing the neoR marker,and then the neoR marker is replaced with another targeting vector containingthe desired mutation. Following selection, the ES cell clones can be checkedby PCR or Southern blot analysis to ascertain that they contain the correctmutation.6. Special Factors to Consider When Making GeneticallyEngineered Mice for Aging Studies6.1. Adult-Specific ExpressionIn aging studies, the researcher is generally interested in the effects of alterationsin a particular gene product in a particular tissue in the adult animal.Many cell-type specific promoters are available that will allow the researcherto target the particular tissue of interest; however, they often allow expressionprior to adulthood in a manner that may confound the researcher’s analysis ofaging effects related to changes in the activity of the gene product. To eliminateany developmental effects of gene alteration, inducible systems can be used.Previously, the only types of inducible systems available were those thatinvolved the use of inducing agents that themselves could be toxic to cells orelicit pleiotrophic changes that could confound the analysis of any resultingphenotype, for example, heat shock, heavy metals, glucocorticoids, and interferon(27–29). These systems were also plagued by low levels of inducibility,leakiness in the noninduced state, and the inability to achieve effective inductionin vivo. In the last few years, new inducible systems have been developedthat overcome many, if not all, of the drawbacks of the earlier systems andallow both temporal and cell specificity of gene expression.The tetracycline (Tet) system allows inducible expression of transgeneswithout the requirement of agents that might themselves cause cellular stress(30). In this system, a Tet-inducible reverse transcriptional transactivator (rTta)protein consisting of a “reverse” Tet repressor (rTetR) fused to the herpes simplexvirus VP16 transcription activation domain is constitutively expressed viaa cytomegalovirus immediate early promoter (pCMV) from what has beentermed the “regulatory” transgene. Expression of this transgene can be limited