Generation of mice carrying the conditional prothrombin knockout allele (fII^lox) and Cre-mediated recombination. (A) Organization of the wild-type prothrombin gene (fII^WT), the fII^lox targeting vector, the modified prothrombin allele carrying LoxP sites (fII^lox), the Cre-disrupted mutant prothrombin allele (fII^CreΔ), and the previously described constitutive prothombin null (fII^null) allele. Exons are indicated as numbered boxes. The position and orientation of diagnostic PCR primers used are indicated with solid triangles. The location and size of HindIII fragments used to identify the fII^lox, fII^CreΔ, and fII^null alleles by Southern blot analysis are indicated by lines below each allele. The relative position of the DNA fragment used as a hybridization probe in Southern blot analyses is highlighted with a solid bar. (B) Southern blot of HindIII-digested liver genomic DNA prepared from individual fII^lox/− mice carrying (Mx⁺) or lacking (Mx⁻) the Cre transgene. Hepatic DNA was isolated from either untreated mice or animals given a single intraperitoneal injection of poly I:C (5 μg/g). Note that near-complete gene recombination was observed in individual Mx⁺fII^lox/− mice 5 days after treatment with poly I:C (lanes 8-10), whereas the floxed allele was largely intact in Cre transgene-negative animals (Mx⁻) regardless of any prior exposure to poly I:C. (C) Complementary multiplex PCR analysis of genomic liver DNA from individual fII^lox/− adults after poly I:C treatment. The PCR products derived from the fII^WT allele, the intact fII^lox allele, and the Cre-recombined fII^lox allele (fII^CreΔ) were 402 bp, 438 bp, and 318 bp, respectively. Note that only trace recombination was observed in Mx⁺fII^lox mice in the absence of poly I:C, whereas the fII^lox allele was largely disrupted after poly I:C treatment in Mx⁺ animals. (D) Northern blot analysis of total liver RNA isolated 5 days after poly I:C administration to Mx⁻ and Mx⁺ fII^lox/− mice illustrating the selective loss of detectable fII mRNA in Mx⁺fII^lox mice. (E) Western blot analysis of plasma prothrombin in unchallenged fII^lox/− adults and animals challenged 5 days previously with poly I:C. Note that plasma prothrombin was approximately 10% to 20% of normal in unchallenged fII^lox/− mice and was reduced to immunologically undetectable levels in poly I:C-treated Mx⁺fII^lox/− mice. H indicates HindIII; B, BamHI; K, KpnI; S, SmaI, E, EcoRI.