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Fig. 1. Strategy for tissue-restricted conditional knockout (CKO) of Cx43. (A) Schematic representation of wild-type, floxed and floxed-out alleles, demonstrating the locations of NcoI sites, the probe used for Southern blotting (solid bar) and primer locations for PCR (small arrows). ORF, open reading frame; UTR, untranslated region. (B,C) Mating strategies for the generation of Wnt1-(B) and P3pro-Cre-(C) mediated CKO mice. Ctrl, control; flox, floxed; fo, floxed-out (Cx43-null). (D) Southern blotting of genomic DNA samples from tails of offspring from P3proCre⁺:Cx43^fo/wt x P3proCre^-:Cx43^flox/wt matings. NcoI-digested genomic DNA yields a 6.5 kb wild-type band, a 5.4 kb floxed band and a 4.3 kb floxed-out band. Samples shown are from P3proCre^-:Cx43^flox/fo (lane 1), P3proCre⁺:Cx43^fo/wt (lane 2), P3proCre⁺:Cx43^flox/fo (Cx43-PCKO; lane 3) and P3proCre^-:Cx43^flox/wt pups (lane 4). (E) PCR samples showing similar genotypes to those in corresponding lanes in D. One primer pair is located entirely within exon 2 and generates a 220 bp PCR band from wild-type DNA and a 180 bp band from the floxed allele. A second primer pair generates a 550 bp product from the floxed-out allele and no product from either wild-type or floxed alleles.

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