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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 4ā€“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 alleleā€”that is, knockout mice. See M. R. Capecchi, 1989, Trends Genet. 5:70.
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