Generation of Fib^AEK mice that express normal levels of a mutant form of fibrinogen with a mutation in the fibrinopeptide sequence of the Aα chain immediately upstream of the thrombin cleavage site. (A) Schematic diagram of the gene targeting strategy for inserting 11 nucleic acid substitutions into the endogenous fibrinogen Aα-chain gene of mouse embryonic stem (ES) cells. Black boxes symbolize gene exons. Arrowheads indicate relative positions of nucleotide primers used for PCR-based genotyping. Note that ES cell clones were screened for incorporation of the Fib AαEK HPRT targeting vector by homologous recombination, and deletion of the HPRT minigene was accomplished by crossing mice carrying the targeted allele to transgenic mice expressing Cre recombinase under the control of the cytomegalovirus (CMV) promoter. (B) Summary of the nucleic acid substitutions, and resulting amino acid changes, for the mutated fibrinogen Aα-chain gene of Fib^AEK mice. Asterisks highlight positions of the nucleotide substitutions. (C) Representative PCR analyses to establish animal genotypes using DNA template from ear biopsies of WT, heterozygous, and homozygous mutant Fib^AEK mice. Primers 5 and 6 were used to amplify a 566-bp fragment, which was subsequently digested with PvuII to yield the diagnostic fragments of 402 and 164 bp. (D) RT-PCR analysis of total hepatic RNA isolated from each of 3 individual WT and Fib^AEK homozygous mice. Primers specific to sequences within exons 1 and 5 were used to generate an 1185-bp PCR product. The product generated from a WT transcript was insensitive to cleavage by PvuII, whereas the product generated from the mutant Fib^AEK transcript was cleaved into expected 1047- and 138-bp fragments. (E) Western blot analysis of plasma (nonreducing conditions) harvested from 4 WT mice, 4 Fib^AEK mice, and 1 Fib^−/− mouse using fibrinogen-directed polyclonal antisera. (F) Coomassie blue-stained sodium dodecyl sulfate polyacrylamide gel (reducing conditions) showing affinity-purified fibrinogen preparations from WT and Fib^AEK mice. The positions of the Aα, Bβ, and γ chains are indicated.