Modification of the fibrinogen γ chain gene. (A) Overall structure of the Fibγ ^390–396A gene-targeting vector, the WT fibrinogen γ chain gene, and the targeted Fibγ ^390–396A allele. Exons are depicted as solid areas, and the introduced nucleotide substitutions and PvuII site are indicated in text boxes as 390–396A and PvuII, respectively. The PvuII fragments that were diagnostic for the WT and targeted alleles by Southern blot assays are indicated by thin lines. Arrowheads indicate the position of diagnostic PCR primers used for detecting homologous recombination events in ES cells. Routine animal genotyping was done using primers 1 and 2 followed by PvuII digestion. (B) Partial nucleotide sequence of exons 9 and exon 10 of the WT and γ ^390–396A genes. Asterisks indicate nucleotides that were mutated in the Fibγ ^390–396A allele. A vertical arrow indicates the position of the exon 9 to 10 splice junction. Amino acids altered by the nucleotide substitutions are in italics. Underlined amino acids indicate the glutamine and lysine that participate in transglutaminase-mediated cross-linking. Amino acids that are known to be critical for platelet integrin receptor α_IIbβ₃ binding (23) are bracketed. (C) Representative PCR analyses to establish animal genotypes using DNA template from ear biopsies of WT, hemizygous, and homozygous mutant Fibγ ^390–396A mice. Primers 1 and 2 were used to amplify a 527-bp fragment, which was subsequently digested with PvuII to yield the diagnostic fragments of 304 bp and 223 bp. ddH₂O, double distilled water.