Figure 5. Inter-loxP distance and site type influence on Cre-mediated recombination efficiency and mosaicism.

(a) Male homozygous floxed mouse strains, with a fixed inter-loxP distance of 6.9 kb at different loci and carrying either wildtype loxP sites or mutant loxP sites (lox71 and lox66), were bred with female Ella-cre, CMV-cre, or Sox2-cre mice. (b) Male homozygous floxed mouse strains, with an inter-loxP distance of 2.7 kb and 2.9 kb at different loci, having wildtype loxP sites and mutant lox71/66 sites, respectively, were bred with female Sox2-cre mice. (c) Male homozygous floxed mouse strains, with an inter-loxP distance of 6.9 kb or 2.9 kb at the same locus and with mutant loxP sites (lox71 and lox66), were bred with female Sox2-cre mice. In Figures 5a, b, and c, F1 offspring were genotyped using PCR from tail DNA to detect floxed, mosaic, and recombined alleles. Offspring with both recombined and floxed alleles were considered mosaic. Note that percentages were rounded to the nearest whole number. (d) Rhbdf1^−/− Rhbdf2^−/− double KO mice exhibit the ‘eyelids open at birth’ phenotype (indicated by arrows) and post-natal lethality⁴⁵. However, when we generated Rhbdf1 conditional KO mice with mutant loxP sites (lox71 and lox66) and an inter-loxP distance of 6.9 kb (Rhbdf1^{flox/flox-6.9kb}), and crossed with Sox2-cre mice, we were unable to generate Rhbdf1^{flox/flox-6.9kb} Rhbdf2^−/− mice carrying the recombined Rhbdf1 allele (top panel—mosaic). Conversely, reducing the inter-loxP distance of the Rhbdf1^{flox/flox-6.9kb} conditional KO allele from 6.9 kb to 2.9 kb using CRISPR/Cas9-mediated deletion of up to 3 kb (Rhbdf1^{flox/flox-2.9kb}), and crossing them with Rhbdf2^−/− and Sox2-cre mice, resulted in the generation of the Rhbdf1 recombined allele. This led to in utero lethality in Rhbdf1^{flox2.9/flox2.9} Rhbdf2^−/− Sox2-cre mice (bottom panel—complete recombination). (e) The ‘eyelids open at birth’ phenotype (indicated by arrows) was observed in the Rhbdf1^{flox/flox-2.9kb} Rhbdf2^−/− K14-cre mice (bottom panel—complete recombination) but not in the keratinocyte-specific knock-out Rhbdf1^{flox/flox-6.9kb} Rhbdf2^−/− K14-cre mice (top panel—mosaic). (f) In utero lethality was only observed with the Rhbdf2^−/− Rhbdf1^{flox/flox-2.9kb} conditional KO allele (bottom panel—complete recombination), not with the Rhbdf2^−/− Rhbdf1^{flox/flox-6.9kb} allele (top panel—Mosaic), when endothelial-specific Cdh5-cre was utilized.