In diploid organisms, as noted in Section 6.1, the phenotypic effect of a recessive allele is expressed only in homozygous individuals, whereas dominant alleles are expressed in heterozygotes. Thus an individual must carry two copies of a recessive allele, but only one copy of a dominant allele, to exhibit the corresponding phenotypes. We have seen how strains of mice that are homozygous for a given recessive knockout mutation can be produced by crossing individuals that are heterozygous for the same knockout mutation (see Figure 6-38). In experiments with cultured animal cells, however, it is usually difficult to disrupt both copies of a gene in order to produce a mutant phenotype. Moreover, the difficulty of producing strains with both copies of a gene mutated is often compounded by the presence of related genes of similar function that must also be inactivated in order to reveal an observable phenotype.

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For certain genes, the difficulties of producing homozygous knockout mutants can be avoided by use of an allele carrying a dominant-negative mutation. These alleles are genetically dominant; that is, they produce a mutant phenotype even in cells carrying a wild-type copy of the gene. However, unlike other types of dominant alleles, dominant-negative alleles produce a phenotype equivalent to that of a loss-of-function mutation.

Useful dominant-negative alleles have been identified for a variety of genes and can be introduced into cultured cells by transfection or into the germ line of mice or other organisms. In both cases, the introduced gene is integrated into the genome by nonhomologous recombination. Such randomly inserted genes are called transgenes; the cells or organisms carrying them are referred to as transgenic. Transgenes carrying a dominant-negative allele are usually engineered so that the allele is controlled by a regulated promoter, which allows expression of the mutant protein in particular tissues or at particular times. As noted above, the random integration of exogenous DNA via nonhomologous recombination occurs at a much higher frequency than insertion via homologous recombination. Therefore, the production of transgenic mice is an efficient and straightforward process (Figure 6-40).

Among the genes that can be functionally inactivated by introduction of a dominant-negative allele are those encoding small (monomeric) GTP-binding proteins belonging to the GTPase superfamily. As we will see in several later chapters, these proteins (e.g., Ras, Rac, and Rab) act as intracellular switches. Conversion of these small GTPases from an inactive GDP-bound state to an active GTP-bound state depends on their interacting with a corresponding guanine nucleotide exchange factor (GEF). A mutant small GTPase that permanently binds to the GEF protein will block conversion of endogenous wild-type small GTPases to the active GTP-bound state, thereby inhibiting them from performing their switching function (Figure 6-41).

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