Fig. 1. Targeting of the TβRI gene. (A) The TβRI wild-type locus, the targeting vector, the targeted allele and the Cre-loxP recombined allele. The targeting vector was generated by inserting a loxP-flanked neomycin (neo) cassette in the Bsu36 site upstream of exon 3 and a single loxP in the NheI site downstream. A thymidine kinase (tk) gene was placed at one end of the construct for negative selection against random integration. Transient expression of the Cre enzyme allowed excision of exon 3 and the neo gene in the targeted allele. Exons are indicated by filled boxes and loxP sites by filled arrows. PCR primers for screening are indicated by open arrows and numbers: 1, RI5′; 2, loxdown; 3, lnl3′; 4, lnl5′; 5, llox3′. Restriction enzymes: Bs, Bsu36; EI, EcoRI; EV, EcoRV; K, KpnI; N, NotI; Nh, NheI. (B) PCR screening for homologous recombination of the targeting vector using the 5′ external primer RI5′ (1) and the vector-specific primer loxdown (2). (C) Genotyping by PCR of an E9.5 litter from a heterozygous intercrossing. The three primers lnl3′ (3), lnl5′ (4) and llox3′ (5) generate specific bands for the wild-type (0.15 kb, primers 3 and 4) and the deleted alleles (0.35 kb, primers 3 and 5). (D) Analysis of TGF-β receptor expression on endothelial cells. Endothelial cells, isolated from wild-type, heterozygous or TβRI^–/– embryos were grown to confluency, affinity labeled with [¹²⁵I]TGF-β1, followed by cross-linking. Cell lysates were subjected to immunoprecipitation using antisera against TβRI or TβRII. (E) Northern blot analysis of total RNA from endothelial cells derived from wild-type and homozygous mutated embryos hybridized with a full-length TβRI cDNA probe. GAPDH mRNA was measured to control for equal loading.