FIG. 1.

(A) Targeting strategy. The structure of the MLC2V locus and the targeting vector are shown first and second, respectively. The mutated locus after homologous recombination is shown third, and the modified locus by Cre recombination is shown at the bottom. ATG is the transcriptional start site. The closed arrowheads represent the loxP sites. B, BamHI; H, HindIII; X, XbaI. (B) Genotyping of ES cell clones after homologous recombination. Genomic DNA was digested with XbaI and analyzed by Southern blotting. The 5′ probe (a BamHI-HindIII fragment) was used. Hybridization with the 5′ probe revealed the expected 5.5- and 6.5-kb fragments from the wild-type and targeted alleles, respectively. (C) Genotyping of ES clones after Cre recombination. Genomic DNA was digested with BamHI and analyzed by Southern blotting. Hand1/eHAND cDNA was used as a probe. The expected 4-kb fragment from the original targeted allele and the 2-kb fragment from the Cre mutated allele were revealed. Fragments from the wild-type allele for Hand1/eHAND were also detected (not shown on this figure). (D, E, and F) Immunohistochemistry with an anti-FLAG antibody. FLAG-tagged Hand1/eHAND protein was expressed in the nuclei of ventricular cells, whereas the expression was not detected in atrial cells in Hand1/eHAND KI embryos (E). FLAG-tagged Hand1/eHAND protein was expressed in the whole ventricle (F). A, atrium; V, ventricle. Bars, 100 μm.