Fig. 1. Generation of Bmp4^S2G mice. (A) Schematic illustration of cleavage patterns of wild-type and S2G mutant forms of proBMP4. (B) Wild-type or mutant forms of the BMP4 precursor were expressed in HEK 293 cells and cleavage products analyzed by probing western blots of cell lysates with anti-HA antibody which recognizes an epitope tag present in the prodomain. Bands corresponding to uncleaved precursor, intact prodomain following cleavage at the S1 site, and intact prodomain following cleavage at the S2 site are indicated. Introduction of a point mutation [BMP4(mS2G)] that prevents cleavage at the S2 site still allows for cleavage at the S1 site whereas both sites are cleaved in native proBMP4. Mature BMP4 cleaved from either precursor migrates with an identical molecular mass (data not shown). (C) Genomic organization of the wild-type Bmp4 allele, the targeting vector and the Bmp4^S2Gneo allele. The positions of the probes used for Southern analysis, and primers for PCR genotyping (arrows) are indicated. (D) Southern blot analysis of genomic DNA from targeted (H6, 2D8 and 2A10) or non-targeted (ES) ES cells. Genomic DNA was digested as indicated and probed with the internal (probe 2) or external (probe 3) genomic fragments shown in panel C. (E) PCR genotyping of progeny generated by interbreeding Bmp4^S2G/+ mice. The PCR product generated by the mutant allele is 293 bp and that generated by the wild-type allele is 259 bp.