Aging Aging

Genetically Engineered Mice 363as the rate of turnover of the protein product produced from it. However, in thelast decade a second type of genetic engineering called gene targeting or gene“knockout” has been developed that allows the elimination of expression of anendogenous gene of interest by producing an insertional mutation in theendogenous gene. Here, a cloned fragment from the gene (a minimum of 2 kbin size) is altered in vitro usually via insertion of a neomycin resistance (neoR)gene into a region of the gene vital for its expression, for example, an exon(15,16). Thymidine kinase sequences from the herpes simplex virus (HSV-tk)are next introduced at both ends of the linearized gene fragment and the alteredgene (termed the “targeting vector”) is introduced into pluripotent embryoderivedstem (ES) cells either via either direct injection or electroporation (fora review of ES culture conditions, see ref. 17). The most well-studied and commonlyused ES cells are derived from the inbred 1295v mouse strain, althoughthe use of ES cells from both inbred C57B1/6 and hybrid C57B1/6 × CBA/JNCrj backgrounds has also been described (18–20).Following introduction of the altered gene fragment into the ES cell, homologousrecombination occurs between it and the endogenous gene in the EScell DNA at the ends of the region of homology between the transgene andgenomic target sequence so that the copy of the gene containing the neoR insertionalmutation replaces the normal copy of the endogenous gene (14,15). Useof DNA in the targeting vector that is derived from the same background strainas the ES cells used may improve the frequency of homologous recombination,that is, syngeneic or isogenic DNA (21).During homologous recombination, the distal HSV-tk sequences are eliminated.When the transgene is inserted into the ES cell DNA via random integration(a more frequent event than homologous recombination in mammaliancells unlike in yeast), the HSV-tk sequences are retained. Cells containing thehomologously integrated copy of the gene can therefore then be selected bygrowth in media containing both neomycin and gancyclovir to select for cellscontaining the neoR insertion but not the HSV-tk sequences (“positive–negativeselection”). Other methods to distinguish homologous recombination fromrandom integration include using targeting vectors containing a positive selectionmarker lacking either its own promoter or polyadenylation site (22).ES cells containing the neoR gene insertion in the gene of interest are next introducedinto mouse blastocysts by either injection into the cavity of a host blastocyst(a 3.5-d embryo at the 32-cell stage, 23) or aggregation with a hostembryo at the morula stage (2.5-d embryo at the 8–16-cell stage, 23–26).Because they remain pluripotent even following long periods of growth in tissueculture, the ES cells will contribute to all tissues of the developing animalincluding cells of the germ line. ES cells derived from mice with one coat colormay be introduced into a host embryo of a different coat color to distinguish