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| Modern Genetic Analysis Chapter 11. Applications of Recombinant DNA Technology Recombinant DNA Technology in Eukaryotes Figure 11-20. Producing a knockout mouse carrying the targeted mutation. (a) Embryonic stem (ES) cells (green at far left) are isolated from an agouti mouse strain and altered to carry a targeted mutation in one chromosome. The ES cells are then inserted into young embryos, one of which is shown. Coat color of the future newborns is a guide to whether the ES cells have survived in the embryo. Hence, ES cells are typically put into embryos that, in the absence of the ES cells, would acquire a totally black coat. Such embryos are obtained from a black strain that lacks the dominant agouti allele. (See Chapter 6.) The embryos containing the ES cells grow to term in surrogate mothers. Newborns with agouti shading intermixed with black indicate that the ES cells have survived and proliferated in an animal. (Such mice are called chimeras because they contain cells derived from two different strains of mice.) Solid black coloring, in contrast, would indicate that the ES cells had perished, and these mice are excluded. A represents agouti, a black; m is the targeted mutation and M is its wild-type allele. (b) Chimeric males are mated to black (nonagouti) females. Progeny are screened for evidence of the targeted mutation (green in inset) in the gene of interest. Direct examination of the genes in the agouti mice reveals which of those animals (boxed) inherited the targeted mutation. Males and females carrying the mutation are mated to one another to produce mice whose cells carry the chosen mutation in both copies of the target gene (inset) and thus lack a functional gene. Such animals (boxed) are identified definitively by direct analyses of their DNA. The knockout results in a curly-tail phenotype. (Redrawn from M. R. Capecchi, "Targeted Gene Replacement." Copyright © 1994 by Scientific American, Inc. All rights reserved.)

© 1999 by W. H. Freeman and Company. All rights reserved.