Cells expressing Cre protein
Cells expressing Cre
Gene function is disrupted
▲ EXPERIMENTAL FIGURE 9-40 The loxP-Cre recombination system can knock out genes in specific cell types. Two loxP sites are inserted on each side of an essential exon (2) of the target gene X (blue) by homologous recombination, producing a loxP mouse. Since the loxP sites are in introns, they do not disrupt the function of X. The Cre mouse carries one gene X knockout allele and an introduced cre gene (orange) from bacteriophage P1 linked to a cell-type-specific promoter (yellow). The cre gene is incorporated into the mouse genome by nonhomologous recombination and does not affect rying a germ-line knockout may have defects in numerous tissues or die before the developmental stage of interest. To address this problem, mouse geneticists have devised a clever technique to inactivate target genes in specific types of somatic cells or at particular times during development.
This technique employs site-specific DNA recombination sites (called loxP sites) and the enzyme Cre that catalyzes recombination between them. The loxP-Cre recombination system is derived from bacteriophage P1, but this site-specific recombination system also functions when placed in mouse cells. An essential feature of this technique is that expression of Cre is controlled by a cell-type-specific promoter. In loxP-Cre mice generated by the procedure depicted in Figure 9-40, inactivation of the gene of interest (X) occurs only in cells in which the promoter controlling the cre gene is active.
An early application of this technique provided strong evidence that a particular neurotransmitter receptor is important for learning and memory. Previous pharmacological and physiological studies had indicated that normal learning requires the NMDA class of glutamate receptors in the hippocampus, a region of the brain. But mice in which the gene encoding an NMDA receptor subunit was knocked out died the function of other genes. In the loxP-Cre mice that result from crossing, Cre protein is produced only in those cells in which the promoter is active. Thus these are the only cells in which recombination between the loxP sites catalyzed by Cre occurs, leading to deletion of exon 2. Since the other allele is a constitutive gene X knockout, deletion between the loxP sites results in complete loss of function of gene X in all cells expressing Cre. By using different promoters, researchers can study the effects of knocking out gene X in various types of cells.
neonatally, precluding analysis of the receptor's role in learning. Following the protocol in Figure 9-40, researchers generated mice in which the receptor subunit gene was inactivated in the hippocampus but expressed in other tissues. These mice survived to adulthood and showed learning and memory defects, confirming a role for these receptors in the ability of mice to encode their experiences into memory.
Dominant-Negative Alleles Can Functionally Inhibit Some Genes
In diploid organisms, as noted in Section 9.1, the phenotypic effect of a recessive allele is expressed only in homozygous individuals, whereas dominant alleles are expressed in heterozygotes. That is, 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 9-39). For 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 in 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.
For certain genes, the difficulties in 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. But unlike other
Inject foreign DNA into one of the pronuclei
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