create clonal line clone "knockout 'mouse clone "knockout 'mouse rise to both somatic tissues and the germline. Once mice are produced thai contain transformed germ cells, matings among siblings are performed to obtain homozygous mutants. Sometimes these mutants must be analyzed as embryos due to lethality. With other genes, the mutant embryos develop into full-grown mice, which are then examined using a variety of techniques.

Mice Exhibit Epigenetic Inheritance

Studies on manipulated mouse embryos led to the discovery of a very peculiar mechanism of non-Mendeiian, or epigenetic, inheritance. This phenomenon is known as parental imprinting (Figure 21-27). The basic idea is that only one of the two alleles for certain genes is active. This is because the other copy is selectively inactivated cither in the developing sperm cell or the developing egg. Consider the case of the Igf-2 gene. It encodes an insulin-like growth factor that is expressed in the gut and liver of developing fetuses. Only the Igf-2 allele inherited from the father is actively expressed in the embryo. The other copy, although perfectly normal in sequence, is inactive. The

FIGURE 21-26 Gene knockout via homologous recombination. The figure outlines the method used to cieate a cell line lacking any given gene. Homologous recombination that occurs within a target gene (shown in green) results tn the incorporation of NEO and disruption of that gene. Nonhomologous, or random, recombination can result in the incorporation of the disrupted gene containing NEO, and the gene encoding thymidine kinase (TK). Clones carrying both constructs survive exposure to neomycin, but the clones also carrying TK are subsequently counterselected by growth in gan cydovtr (GANC). Clones containing the construct carrying the target gene with the NEO insertion are thus the only survivors. Once produced, these cells can be cloned and used to generate a complete mouse lacking that same gene (see Figure 21 -24).

Figure 21-27 Imprinting in the mouse. The permanent silencing of one allele of a given gene in a mouse As outlined in the text, and described in detail in Chapter 17, imprinting ensures that only one copy of the mouse !q!2 gene is expressed in each cell. It is always the copy earned on the paternal chromosome (hat is expressed.

somatic cell somatic cell l__I

meiosis removes imprinting

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