Targeted disruption of the Pros gene. (A) A 23.7-kb targeting vector was constructed that contains a single loxP site 5′ of Pros exon 3, and an Frt-neo-Frt-loxP cassette 3′ of exon 7 (loxP and Frt sites are depicted by red and blue triangles, respectively). Restriction sites StuI (S) and DraIII (D) were introduced at these sites for genotyping purposes. The long 5′ homology arm was 4 kb, and the short 3′ homology arm was 2 kb. Homologous recombination in 129Sv ES cells resulted in a floxNeo allele that was detected by long-range PCR and Southern blotting analysis, as described in supplemental Figure 1. One correctly targeted ES cell clone was injected into blastocysts of C57BL/6J mice to generate chimeras, which in turn transmitted the floxNeo allele to F1 Pros^floxNeo/+ mice. These mice were genotyped by PCR (see supplemental Figure 1). Cre-mediated recombination through crossing of Pros^floxNeo/+ mice to a general Cre deleter strain (Nes-Cre1) resulted in the generation of a null (−) allele. Germline transmission of this null allele yielded Pros^+/− mice in a mixed 129Sv-C57BL/6J genetic background. (B) WT, Pros^+/−, and Pros^−/− mice were genotyped by Southern blotting. Genomic tail DNA of E17.5 embryos from the 3 genotypes were digested by StuI and hybridized with the 3′DraIII probe (Figure 1A). The WT band was 12.2 kb, and the null (−) band was 4.4 kb (arrows). (C) WT, Pros^+/−, and Pros^−/− mice were genotyped by 2 separate PCRs with mouse tail genomic DNA as a template and primer couples PROS-Fwd/PROS-Rv (F and R, respectively [A]) for the WT band (234 pb), and PROS-Fwd/PROSnull-Rv (F and Rnull, respectively [A]) for the null band (571 pb). (D) Reduced levels of PS in the plasma of Pros^+/− mice compared with WT mice were demonstrated after polyacrylamide gel electrophoresis of nonreduced plasma samples by Western blotting with a rabbit polyclonal antibody raised against mouse PS. Mouse plasma PS migrated at approximately 80 kDa (arrow).