Somatic Cell Recombination Can Inactivate Genes in Specific Tissues
Investigators are often interested in examining the effects of knockout mutations in a particular tissue of the mouse, at a specific stage in development, or both. However, mice carrying 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 6-39, inactivation of the gene of interest (X) occurs only in cells in which the promoter controlling the cre gene is active.
EXPERIMENTAL FIGURE 6-39 The loxP-Cre recombination system can knock out genes in specific cell types. A loxP site (purple) is inserted on each side of an essential exon (exon 2) of the target gene (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. A 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 the function of other genes. In the loxP-Cre mice that result from crossing these two types of mice, 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.
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 neonatally, precluding analysis of the receptor’s role in learning. Following the protocol in Figure 6-39, 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.