Figure 1. Generation of mCx47M282T mice with LacZ reporter gene.

A, Scheme of homologous recombination of the targeting vector into the Cx47 coding region. The resulting transgenic allele (Cx47M282Tneo) includes mCx47M282T coding DNA, an internal ribosome entry site (IRES) followed by a nuclear localization signal (nls) fused to LacZ coding DNA and a neomycin selection cassette flanked by frt-sites. The endonuclease BsmBI restriction site was generated by T to C transition on nucleotide 845 of the Gjc2 coding region, resulting in the mutant mCx47M282T. B, Flp recombinase activity causes deletion of the neomycin selection cassette. Specific primer binding sites and MfeI restriction sites are indicated. C, PCR products specific for wild-type (570 bp) and transgenic loci (730 bp) using DNA obtained from tail tip tissue. D, Presence of the mutant allele was proven by PCR amplification of a 700 bp Cx47 fragment and subsequent BsmBI digestion. Only mutant alleles yielded the 350 bp fragment. E, PCR analysis resulted in amplification of the 1,700 bp fragment of the neomycin selection cassette deprived allele and the 450 bp fragment of the Cx47M282Tneo locus. F, Homologous recombination and different allelic combinations were confirmed by Southern blot hybridization using a probe derived from the 3′ region of Cx47. MfeI digestion of genomic DNA prepared from liver resulted in the of 4.6 kb fragment for the Cx47 wildtype allele, the 10.5 kb fragment for the Cx47M282Tneo allele and the 8.5 kb fragment for the Cx47M282T allele.