GP VI gene structure and targeted disruption. (A) The mouse GP VI gene is schematically presented as it spans a portion of GenBank Accession no. AC087129.1. Based on an alignment of the mouse GP VI cDNA sequence and genomic sequence, 8 exons (▦) and introns were identified. By sequence alignment with the human GP VI cDNA, a putative initiating Met codon (ATG) is shown in exon 1. (B) An expanded region of the wild-type mouse GP VI gene containing exons 1 to 3 is shown. (C) A 9-kb HindIII restriction fragment spanning exons 1 to 3 was subcloned into a pBS/KS-vector and altered via site-directed mutagenesis to place a stop codon (TGA) immediately 3′ to the initiating Met codon followed by an XhoI restriction site. Following successful mutagenesis a neocassette was cloned within the exon 1 sequence using the restriction site created during mutagenesis. (D) Successful homologous recombination in mouse ES cells results in the replacement of a 7-kb HincII restriction fragment with an 8.7-kb HincII fragment. (E) Germ line transmission of the ES cell genotype was obtained producing GP VI^het mice. The breeding of GP VI^het mice produced the expected 3 genotypes and shown is a representative Southern blot of each of the genotypes revealing the wild-type GP VI gene (7 kb) and the targeted allele (8.7 kb). (F) A mouse monoclonal antibody recognizing both human and mouse GP VI proteins was used to verify the lack of the GP VI polypeptide in platelet lysates from GP VI^null mice. Shown is a Western blot of platelet lysates from each of the 3 genotypes. A gene dosage effect of GP VI levels is seen as a consequence of the altered GP VI allele. (G) The same filter was reprobed with an anti–14-3-3ζ polyclonal antibody to confirm the presence of platelet proteins in each lane.