Fig. 1. Comparison among members of the Mesp-related gene family (A) and strategy of gene replacement of mouse Mesp2 with zebrafish mespb (B,C). (A) Comparison of amino acid sequences in the bHLH motif. The percentage homology to Mesp2 within and outside the bHLH motif are shown. Differences are indicated in green (Mespb) and purple (Mesp1). (B) Knock-in strategy. The top line shows the genomic organization of the Mesp2 gene; the second shows the structure of the targeting vector; the third is the predicted structure of the Mesp2 locus following homologous recombination. Mesp2 exons (pink boxes) were completely deleted and replaced with the zebrafish mespb cording region flanked with floxed neo cassette and poly(A) signal (the arrowheads on the line represent loxP sites). Chimeric mice generated from recombinant ES cells containing targeted allele, Mesp2^neo–mespb, were mated with CAG-Cre mice to excise the floxed neo cassette, resulting in the generation of the Mesp2^mespb allele. The probe used for Southern blot analysis is indicated. Restriction enzymes: B, BamHI; E, EcoRI; Hi, HincII; H, HindIII; K, KpnI; P, PstI; S, SacI; Sm, SmaI; X, XbaI. Arrows indicate PCR primers. (C) Genomic Southern blot analysis of SacI-digested DNA from embryos with various Mesp2 alleles. Arrowheads show the 6.0 kb fragment of the wild-type allele, the 2.3 kb targeted Mesp2^neo–mespb, and the 4.7 kb Cre-excised Mesp2^mespb allele. Genotypes of progeny are indicated at the top of each lane. All represent genotypes of the Mesp2 allele.