EXPERIMENTAL FIGURE 6-37 Isolation of mouse ES cells with a gene-targeted disruption is the first stage in production of knockout mice. (a) When a recombinant DNA construct is introduced into embryonic stem (ES) cells, random insertion via nonhomologous recombination occurs much more frequently than gene-targeted insertion via homologous recombination. Recombinant cells in which one allele of gene X (orange and white) is disrupted can be obtained by using a recombinant vector that carries gene X disrupted with neo^r (green), which confers resistance to G-418, and, outside the region of homology, tk^HSV (yellow), the thymidine kinase gene from herpes simplex virus. The viral thymidine kinase, unlike the endogenous mouse enzyme, can convert the nucleotide analog ganciclovir into the monophosphate form; this is then modified to the triphosphate form, which inhibits cellular DNA replication in ES cells. Thus ganciclovir is cytotoxic for recombinant ES cells carrying the tk^HSV gene. Nonhomologous insertion includes the tk^HSV gene, whereas homologous insertion does not; therefore, only cells with nonhomologous insertion are sensitive to ganciclovir. (b) Recombinant cells are selected by treatment with G-418, since cells that fail to pick up the construct or integrate it into their genome are sensitive to this cytotoxic compound. The surviving recombinant cells are treated with ganciclovir. Only cells with a targeted disruption in gene X, and therefore lacking the tk^HSV gene and its accompanying cytotoxicity, will survive. See S. L. Mansour et al., 1988, Nature 336:348.