Fig. 1. Generation of Smad1-deficient mice. (A,B) Targeting strategies. Thick lines represent Southern probes and arrows the direction of transcription. (A) The Smad1^Robm1 (m) vector replaces most of the first coding exon (yellow box) and 30 bp of 3' flanking sequence with a PGK-neo cassette. (B) The Smad1^RobPC vector introduces an EcoRI (R) and loxP site (green triangle) 2 kb 5' of the first coding exon and a loxP-flanked (blue and red triangles) hygro cassette 1.7 kb 3' of the exon. Germline Cre-mediated excision of the Smad1^RobPC allele was used to produce the Smad1^Robcn (cn) allele, which retains part of the 5' and 3' loxP site (red/green triangle). (C) Southern blot analysis of EcoRI-digested DNA from m/+ intercross embryos using the 5' external probe. (D) Southern blot analysis of BamHI-digested DNA from cn/+ intercross embryos using the 5' internal probe. (E) Northern blot analysis of RNA from wild-type (+/+), Smad1 heterozygous (m/+, cn/+) or Smad1-deficient (m/m, cn/cn) 9.5 dpc embryos. A Smad1 probe detects a 2.8 kb wild-type and a 2.2 kb mutant band. (F) Western blot analysis of individual 9.5 dpc embryos and as a control, COS cells mock transfected or transfected with an human Smad1 expression construct and probed with an antibody that recognizes the MH2 domain (amino acids 147-258) of Smad1, Smad5 and Smad8. This antibody is predicted to recognize potentially truncated Smad1 proteins produced from either Smad1 allele. A single band (asterisk) of approximately 55 kDa corresponds to full-length Smad1. No intact or truncated products are detected in homozygous embryos. H, HindIII; M, MluI; S, StuI; B, BamHI; P, PstI; R, EcoRI.