Specific Genes Can Be Permanently Inactivated in the Germ Line of Mice

Many of the methods for disrupting genes in yeast can be applied to the genes of higher eukaryotes. These altered genes can be introduced into the germ line via homologous recombination to produce animals with a gene knockout, or simply “knockout.” Knockout mice in which a specific gene is disrupted are powerful experimental systems for studying mammalian development, behavior, and physiology. They are also useful for studying the molecular basis of certain human genetic diseases.

Knockout mice are generated by a two-stage procedure. In the first stage, a DNA construct containing a disrupted allele of a particular target gene is introduced into embryonic stem (ES) cells. These cells, which are derived from the blastocyst, can be grown in culture through many generations (see Figure 21-7). In a small fraction of transfected cells, the introduced DNA undergoes homologous recombination with the target gene, although recombination at nonhomologous chromosomal sites occurs much more frequently. To enable selection for cells in which homologous recombination and gene-targeted insertion occurs, the recombinant DNA construct introduced into ES cells includes two selectable marker genes (Figure 6-37). One of these genes (neor), which confers resistance to G-418, is inserted within the target gene (X), thereby disrupting it. The other selectable gene, the thymidine kinase gene from herpes simplex virus (tkHSV), is inserted into the construct outside the target-gene sequence. ES cells that undergo recombination between the recombinant DNA construct and the homologous site on the chromosome will contain neor but will not incorporate tkHSV. Because tkHSV confers sensitivity to the cytotoxic nucleotide analog ganciclovir, the desired recombinant ES cells can be selected by their ability to survive in the presence of both G-418 and ganciclovir. In these cells, one allele of gene X will be disrupted.

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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 neor (green), which confers resistance to G-418, and, outside the region of homology, tkHSV (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 tkHSV gene. Nonhomologous insertion includes the tkHSV 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 tkHSV gene and its accompanying cytotoxicity, will survive. See S. L. Mansour et al., 1988, Nature 336:348.

In the second stage in the production of knockout mice, ES cells heterozygous for a knockout mutation in gene X are injected into a recipient wild-type mouse blastocyst, which subsequently is transferred into a pseudopregnant female mouse (Figure 6-38). The resulting progeny will be chimeras, containing tissues derived from both the transplanted ES cells and the host cells. If the ES cells are also homozygous for a visible marker trait (e.g., coat color), then chimeric progeny in which the ES cells have survived and proliferated can be identified easily. Chimeric mice are then mated with mice that are homozygous for another allele of the marker trait to determine if the knockout mutation has been incorporated into the germ line. Finally, mating of mice, each heterozygous for the knockout allele, will produce progeny homozygous for the knockout mutation.

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EXPERIMENTAL FIGURE 6-38 ES cells heterozygous for a disrupted gene are used to produce knockout mice. Step 1: Embryonic stem (ES) cells heterozygous for a knockout mutation in a gene of interest (X) and homozygous for a dominant allele of a marker gene (here, brown coat color, A) are transplanted into the blastocoel cavity of 4.5-day blastocysts that are homozygous for a recessive allele of the marker (here, black coat color, a). Step 2: The early embryos are then implanted into a pseudopregnant female. Those progeny containing ES-derived cells are chimeras, as indicated by their mixed black and brown coats. Step 3: Chimeric mice are then backcrossed to black mice; brown progeny from this mating have ES-derived cells in their germ line. Steps 46: Analysis of DNA isolated from a small amount of tail tissue can identify brown mice heterozygous for the knockout allele. Intercrossing of these mice produces some individuals homozygous for the disrupted allele—that is, knockout mice. See M. R. Capecchi, 1989, Trends Genet. 5:70.

The development of knockout mice that mimic certain human diseases can be illustrated by cystic fibrosis. By the methods discussed in Section 6.4, the recessive mutation that causes this disease was shown to be located in a gene known as CFTR, which encodes a chloride channel. Using the cloned wild-type human CFTR gene, researchers isolated the homologous mouse gene and subsequently introduced mutations in it. The gene-knockout technique was then used to produce homozygous mutant mice, which showed symptoms (i.e., a phenotype), including disturbances to the functioning of epithelial cells, similar to those of humans with cystic fibrosis. These knockout mice are currently being used as a model system for studying this genetic disease and developing effective therapies.