Generation of PROC knock-in hemophilia mice. (A) A schematic of the PROC knock-in strategy. The WT mouse Proc allele with exon 2 to 9 is shown at top, which has the start codon and stop codon, respectively. The targeting vector contains human PROC coding sequence and a downstream neomycin (neo), flanked by LoxP sites, for positive selection. The diphtheria toxin A gene (DTA) was used for negative selection. Once PROC was knocked in, the neo was deleted via Cre-mediated recombination. (B) Breeding strategy to generate PROC^+/+;F8^−/− and PROC^+/+;F9^−/− mice. PROC^+/+ mice were crossed with mice lacking factor VIII (F8^−/−) or factor IX (F9^−/−). (C-F) Quantitative reverse transcription PCR analysis of the mRNA expression. Total mRNA was isolated from mice liver tissues. Results were normalized to GAPDH. Bars represent the means and standard errors of 15 biological replicates (5 mice per group and 3 samples per mouse). Liver GAPDH is set as 100%. Statistical significance was determined between WT and the other groups. ***P < .001; ns, P > .05 (1-way ANOVA Test). (G) Human protein C concentrations in the plasma of various mouse models and human were measured using an ELISA kit for human protein C antigen. Data were expressed as mean ± SD. n = 5 individuals. ELISA, enzyme-linked immunosorbent assay; mRNA, messenger RNA; ns, not significant; UTR, sequence corresponding to the untranslated region on mouse protein C mRNA.