H

loxP site

Cre recombinase

Figure 4 Strategy for Cre recombinase-mediated gene inactivation, A targeting construct is assembled from DNA segments AB, BC and CD of the murine target gene locus (wild-type allele). Essential exon sequences in fragment BC (filled box) are flanked by introns (solid line) containing the neo expression cassette and two loxP sites (arrowheads). Integration of the targeting vector into the wild-type allele does not affect target gene expression because only noncoding intron sequences are modified (floxed allele). However, Cre recombinase activity will cause site-specific excision of exon sequences flanked by the two loxP sites, disrupting the target gene locus (disrupted allele).

Mouse with lloxed target gene

Mouse expressing Cre recombinase in T cells

Mouse with lloxed target gene

Exon flanked by loxP sites

Mouse expressing Cre recombinase in T cells

Exon flanked by loxP sites

Mouse wilhflSTie defect m T cells

Figure 5 Tissue-specific gene disruption in mice. A mouse carrying the floxed target gene in all tissues is bred with an animal expressing the Cre recombinase gene under tissue-specific promoter elements (e.g. T cells). In the offspring, activity of Cre recombinase will disrupt the target gene in T cells exclusively, leaving all other cell types unaffected.

possibility to perform a gene knockout experiment in ES cell cultures and to introduce the genetic alteration into mice by transferring the genetically modified ES cells into mouse embryos (Figure 2).

In detail, ES cell clones heterozygous for a gene disruption are isolated by positive/negative selection and are injected into the blastocoel cavity of mouse embryos at day 3.5 of gestation using micromanipulation. These blastocysts are subsequently transferred into pseudopregnant foster mothers to develop to term. The chimeric animals obtained are bred to normal mice and their offspring are tested for the presence of the mutant allele that has been transmitted through the germ line. F1 generation animals which are heterozygous for the gene alteration can then be bred to each other to derive mice homozygous for the mutant allele (Figure 2).

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