TFPI gene-disruption in mice. (A) TFPI replacement construct and partial restriction map of the endogenous locus. Exon 4 encodes the Kunitz-1 domain of mouse TFPI. neo, neomycin phosphotransferase gene; tk, thymidine kinase gene; Bst, Bst1107I; probe, TFPI-specific probe used in Southern analysis. (B) Southern blot confirmation of TFPI gene disruption. The probe indicated in (A) was used to probe Sac I digests of tail DNA from mutant and nonmutant mice (genotype listed). This probe detects DNA fragments of 12 and 4 kb in the wild-type allele and fragments of 7.5 and 4 kb in the disrupted allele. (C) RT-PCR demonstration of the production of two TFPI mRNAs in TFPI_K1(+/−) mice. One PCR product (620 bp) encodes wild-type TFPI, the other (420 bp) encodes TFPI_desK1 , an altered form of TFPI in which the first Kunitz domain (encoded by exon 4 of the TFPI gene) has been deleted. (D) Western blot analysis of plasma TFPI in wild-type and mutant mice (genotype listed). The protein band at 43,000 MW represents wild-type TFPI; an additional form of TFPI (TFPI_desK1 ) at 37,000 MW is apparent in the plasma from TFPI_K1(+/−) mice and a TFPI_K1(−/−) mouse.