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ES cells with targeted disruption in gene X

ES-cell DNA

Inject ES cells into blastocoel cavity of early embryos

4.5-day blastocyst

Inject ES cells into blastocoel cavity of early embryos

4.5-day blastocyst

Surgically transfer embryos into pseudopregnant female

Foster mother

Possible progeny

Foster mother

Chimeric

Possible progeny

Black

Select chimeric mice for crosses to wild-type black mice

Select chimeric mice for crosses to wild-type black mice

Possible germ cells: All germ cells: A/X+; A/X-; a/X* a/X+

ES cell-derived progeny will be brown

Progeny from ES cell-derived germ cells

Screen brown progeny DNA to identify X/X+ heterozygotes

Mate X /X* heterozygotes

Screen progeny DNA to identify X /X- homozygotes

Knockout mouse confers G-418 resistance, is inserted within the target gene (X), thereby disrupting it. The other selectable gene, the thymidine kinase gene from herpes simplex virus (tkHSV) , confers sensitivity to ganciclovir, a cytotoxic nucleotide analog; it is inserted into the construct outside the target-gene sequence. Only ES cells that undergo homologous recombination can survive in the presence of both G-418 and ganciclovir. In these cells one allele of gene X will be disrupted.

< EXPERIMENTAL FIGURE 9-39 ES cells heterozygous for a disrupted gene are used to produce gene-targeted 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 embryos that are homozygous for a recessive allele of the marker (here, black coat color, a). Step 2|: The early embryos then are implanted into a pseudopregnant female. Those progeny containing ES-derived cells are chimeras, indicated by their mixed black and brown coats. Step 3: Chimeric mice then are backcrossed to black mice; brown progeny from this mating have ES-derived cells in their germ line. Steps 4|- 6: 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. [Adapted from M. R. Capecchi, 1989, Trends Genet. 5:70.]

In the second stage in 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 surrogate pseudopregnant female mouse (Figure 9-39). The resulting progeny will be chimeras, containing tissues derived from both the transplanted ES cells and the host cells. If the ES cells also are homozygous for a visible marker trait (e.g., coat color), then chimeric progeny in which the ES cells survived and proliferated can be identified easily. Chimeric mice are then mated with mice homozygous for another allele of the marker trait to determine if the knockout mutation is incorporated into the germ line. Finally, mating of mice, each heterozygous for the knockout allele, will produce progeny homozygous for the knockout mutation.

Development of knockout mice that mimic certain human diseases can be illustrated by cystic fibrosis. By methods discussed in Section 9.6, the recessive mutation that causes this disease eventually 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. I

Somatic Cell Recombination Can Inactivate Genes in Specific Tissues

Investigators often are interested in examining the effects of knockout mutations in a particular tissue of the mouse, at a specific stage in development, or both. However, mice car-

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