Conditional gene disruption the second generation knockouts

Conventional gene disruption by homologous recombination manifests itself in all cells of an organism and at all stages of ontogenesis. Under certain circumstances however, it might be desirable to induce a gene defect only at distinct stages in development or in a tissue-specific manner. For instance, embryonic lethality caused by disruption of develop-mentally essential genes precludes the analysis of the gene defects in adult animals. One approach to achieve conditional gene inactivation is based on the activity of the Cre recombinase of the bacteriophage PI, a site-specific recombinase which specifically binds to a 34 bp loxP recognition sequence (Figure 3). If two loxP sites are located in the same orientation on a DNA molecule, Cre recombinase-mediated recombination will result in excision of the intervening DNA region, leaving a single loxP site behind. This system has been adapted to achieve inducible gene disruption in mammalian cells.

One such system is described in Figure 4. A targeting construct is generated that contains a region of the target gene modified by integration of the neo marker and one loxP site in one intron and a second loxP site in an adjacent intron. The modified introns flank an essential exon of the target gene. Using standard protocols, this modification is introduced into the target gene of ES cells via homologous recombination (Floxed allele), and mice carrying two copies of the loxP-modified gene are derived (Figure 2). These animals are phenotypically normal as integration of neo and the loxP sites in the intron sequences most likely does not interfere with the functional expression of the gene. However, induced expression of the Cre recombinase in these mice will result in excision of the exon sequences flanked by the two loxP sites and in disruption of the gene (Figure 4, Disrupted allele).

Controlling the temporal and spatial expression of the Cre recombinase in loxP-modified mice is key to determining the conditions of the gene inactivation. Tissue-specific gene inactivation can be achieved by crossing loxP-modified mice with transgenic mice carrying the Cre recombinase gene under the control of tissue-specific promoter elements. In the double transgenic animals, the target gene is inactivated in cell types expressing Cre recombinase but not in other tissues (Figure 5). This approach has been used by Rajewsky and coworkers to inactivate the gene for DNA polymerase (3 in T cells exclusively, and to study the importance of the gene product in the somatic gene rearrangements at the T cell receptor gene locus. Previously, these studies have not been possible because the obligate DNA polymerase (3 gene knockout resulted in embryonic lethality.

See also: Gene therapy; Herpes simplex virus, infection and immunity; Immunodeficiency, animal models; T cell receptor, <*p; T cell receptor, 78; Transgenic animals.

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