Fig 2. Generation of the Impad1 and Clcn7 mouse lines.

(A) Schematic drawing of the wild-type and targeted loci for Impad1. EcoRV restriction sites and fragments for Southern blot analyses are indicated in light blue. (B) Schematic drawings of the wild-type and targeted loci for Clcn7. EcoRI restriction sites and fragments for Southern blot analyses are indicated in light blue. Exons, indicated by arrowheads, are not numbered to simplify the figure. (C) Long range PCR analysis for correct homologous recombination of ES clones with primers SY08.19 and LRPCRneo1. The expected fragment of 7.1 kb confirms correct homologous recombination in the Impad1 locus. (D) Southern blot analysis of Impad1 targeted ES cells after EcoRV digestion using a neomycin specific probe; the 15.2 kb fragment corresponding to correct homologous recombination is indicated. (E) Genotyping PCR analysis for heterozygous Impad1 floxed pups using primers SY08.20 and SY08.21 flanking the lox71 site. The wild-type and floxed allele result in PCR products of 459 bp and 506 bp, respectively. (F) Long range PCR analysis for correct homologous recombination of ES clones with primers SY03.9 and LRPCRneo1. The expected amplicon of 5.8 kb confirms correct homologous recombination in the Clcn7 locus. (G) Southern blot analysis of Clcn7 targeted ES cells after EcoRI digestion using a neomycin specific probe; the 11.9 kb fragment corresponding to correct homologous recombination is indicated. (H) Genotyping PCR analysis for heterozygous Clcn7 floxed pups using primers SY03.11 and SY03.12 flanking the lox71 site. The wild-type and floxed allele result in PCR products of 539 bp and 588 bp, respectively. Black arrowhead: exon; green arrowhead: FRT site; light blue arrowhead: lox71 or loxKR3 site; red arrowhead: targeted exon 2 in Impad1 or targeted exon 7 in Clcn7; green arrow: neomycin cassette; SA: short arm; LA: long arm; E#: exon number; MW: DNA molecular weight marker.