Fig. 1. Generation of Smad4^CA and Smad4^N alleles. (A) Strategy used to flank the first coding exon of Smad4 with loxP sites (red triangles). Targeted clones were identified using a 3' external probe (red line) and an internal probe (blue line). A, ApaI; B, BstZ17I; R, EcoRV; St, StuI; X, XbaI; Xh, XhoI. (B) Southern blot analysis of drug-resistant ES clone DNA digested with BstZ17I and screened using the 3' external probe yields 9 kb wild-type (WT) and 11.2 kb targeted (TA) alleles. (C) Smad4^TA/+ ES cell clones were transiently transfected with Cre recombinase, and DNA from resultant clones was digested with XbaI and screened by Southern blot using the internal probe. Rearrangement of the Smad4^TA allele generates both Smad4 conditional (Smad4^CA) and null (Smad4^N) alleles. (D) PCR analysis of DNA samples from wild-type, Smad4^N/+, Smad4^CA/+ and Smad4^CA/CA homozygous mice. PCR primer locations are indicated in A.