Generation and characterization of prothrombin W215A/E217A gene–targeted mice. (A) Structure of the prothrombin W215A/E217A (fII^WE) gene–targeting vector, the wild-type prothrombin gene, and the targeted fII^WE allele. The positions of the nucleotide substitutions that result in the introduction of the W215A/E217A mutations and a unique PvuII endonuclease site are indicated. Brackets indicate the position of SacI fragments diagnostic by Southern blot analysis for wild-type and fII^WE alleles. The region of the prothrombin gene used as a hybridization probe is highlighted with a thick line labeled “SB probe.” Arrowheads indicate the position of PCR primers used to detect and distinguish the mutant and wild-type alleles. Partial nucleotide sequence of exon 14 of the wild-type and fII^WE alleles are indicated. Asterisks highlight the nucleotides that were mutated within the fII^WE allele and the mutated amino acids are indicated in bold. (B) Southern blot analysis of genomic DNA from wild-type, heterozygous fII^WT/WE, and homozygous fII^WE/WE mice digested with SacI. DNA fragments of 6.2 and 9.9 kb were detected for the wild-type and fII^WE alleles, respectively. (C) Representative PCR analyses of genomic DNA from each of 2 individual wild-type, fII^WT/WE, and fII^WE/WE mice. Primers G1 and G3 produced a 567-bp product specific to the wild-type allele, and primers G1 and HPRT1 produced a 621-bp product specific to the fII^WE targeted allele (top panel). Primers G1 and G2 coupled with a PvuII digest produced a 375-bp product specific to the wild-type allele and 202- and 173-bp products specific to the targeted fII^WE allele (bottom panel). (D) RT-PCR analysis of total hepatic RNA isolated from each of 4 individual wild-type and fII^WT/WE mice. Primers specific to sequences within exons 12 and 14 were used to generate a 266-bp PCR product (top panel). The product generated from a wild-type transcript was insensitive to cleavage by PvuII, whereas the 266-bp product generated from the mutant fII^WE transcript was cleaved into fragments that were 215 and 51 bp in length. Primers specific to the cDNA of β-actin were used in control reaction mixtures for each RT product (bottom panel). (E) Western blot analysis of plasma from wild-type, fII^WT/WE, and fII^WE/WE mice using a polyclonal anti-prothrombin antibody. Comparable levels of prothrombin were observed in 0.5-μL aliquots of citrate plasma collected from 3 individual adult wild-type and 2 individual adult fII^WT/WE mice (top panel). Comparable levels of prothrombin were observed in plasma samples (30 μg of total plasma protein per lane) collected from wild-type, fII^WT/WE, and fII^WE/WE day 1 neonates that were viable and free from overt hemorrhage (bottom panel).