Genotyping of Cre-lox mice and detection of tissue-specific recombination by multiplex PCR.

Conditional gene targeting, based on Cre-lox or other systems, requires frequent genotyping of transgenic mouse populations and monitoring of tissue-specific Cre recombinatory efficiency. This is currently achieved by Southern analysis from tail- and tissue-derived DNA. Multiplex PCR amplification of the floxed (flanked by loxP sites) genomic region, combined with the PCR detection of the Cre transgene, simplifies this task. Here, we show that complete genotyping of a floxed locus is possible with three appropriately placed primers and that this triplex PCR can be performed simultaneously with a universal PCR assay for the detection of Cre transgenes. Using this approach, we also determined the ratios of recombined versus non-recombined floxed genomic segments in genomic DNA samples. This allowed us to estimate the efficiency of in vivo conditional inactivation from biopsy material and tissue samples that were too small for Southern analysis. As many new conditional knockouts are spatiotemporally restricted, such assays will become increasingly useful. The proposed PCR strategy is flexible and may be adapted to the structural specificities of any target gene.

Experimental IGF-I receptor deficiency generates a sexually dimorphic pattern of organ-specific growth deficits in mice, affecting fat tissue in particular.

Reduced IGF type I receptor levels diminish postnatal growth rate and adult body weight in mice. Here, we studied the impact of experimental IGF receptor deficiency on tissue-specific growth by Cre-lox-mediated dosage of a floxed IGF-IR gene. We generated mice with a wide spectrum of receptor deficiency (5-82%), and separated them into two groups with either strong (> or =50%) IGF-IR deficiency (XS mice) or moderate deficiency (<50%, M mice). The growth of XS mice was significantly retarded from 3 wk after birth onward, with respect to M littermates. This effect was twice as strong in males as in females. Growth deficits persisted throughout adult life, and at 10-12 months, most organs and tissues showed specific weight defects. Skin, bone and connective tissue, muscle, spleen, heart, lung, and brain were the most severely affected organs in the XS males. With the exception of muscle and spleen, the same tissues were also significantly reduced in size in females, although to a lesser extent. The most severe growth defect, however, concerned adipose tissue. Fat pad size in XS males was only 29% (females, 44%) of M mice. The estimated number of adipocytes in XS male fat pads was only 21% that of M males (XS female, 27%). Lipid content per cell was significantly higher in XS adipocytes, whereas plasma glucose and insulin levels were low in XS males. Thus, IGF type I receptor deficiency produced mice with disproportionate postnatal organ growth, and these effects depended strongly on sex. A marked reduction in IGF-IR levels resulted in a major defect in adipose tissue.

Cre-mediated germline mosaicism: a method allowing rapid generation of several alleles of a target gene.

Conditional gene targeting uses the insertion of expression cassettes for the selection of targeted embryonic stem cells. The presence of these cassettes in the final targeted chromosomal locus may affect the normal expression of the targeted gene and produce interesting knock down phenotypes. We show here that the selection cassette may then be selectively removed in vivo, using three appropriately positioned loxP sites in the targeted gene and the transgenic mouse EIIaCre. This strategy was applied to two different target genes and we demonstrated that it is reliable and reproducible. First, we generated double transgenic EIIaCre/loxP mice (F1) that showed variable degrees of mosaicism for partially CRE-recombined floxed alleles. Efficiency of EIIaCre at creating mosaicism was dependent on the target gene and on parental transmission of the transgene. The segregation of partially recombined alleles and EIIaCre transgene was obtained in the next generation using mosaic F1 males. Mosaic females were unsuitable for this purpose because they systematically generated complete excisions during oogenesis. Our strategy is applicable to other approaches based on three loxP sites. As this procedure allows generation of knock down (presence of neo), knockout (total exision of the loxP-flanked sequences) and floxed substrains (excision of the selection cassette) from a single, targeted germline mutation and in a single experiment, its use may become more widespread in conditional mutagenesis.