Figure 1.

Targeted deletion of the mouse GP Ibα gene. (A) A 9-kb region of the mouse GP Ibα locus was characterized by restriction enzyme (B = BamHI, H3 = HindIII, RI = EcoRI) and DNA sequence analysis (14, 15). The nucleotide sequence was determined for a 5.4-kb region spanning 2.4 kb 5′ to exon I and 1.7 kb 3′ to exon II (exons indicated as boxes, with the ORF highlighted as a black box in the wild-type allele). A targeting construct was generated by an exact replacement of the GP Ibα ORF with a phosphoglycerate kinase-neo^r cassette (neo^r). A phosphoglycerate kinase-thymidine kinase gene cassette was inserted 3′ to the GP Ibα gene for selection against random integration using gancyclovir. A successfully targeted GP Ibα locus contains a 6.7-kb HindIII restriction fragment as compared with a 9-kb fragment in the wild-type locus. (B) ES cells with a targeted GP Ibα locus were identified by Southern hybridization and injected into blastocysts to generate chimeric mice. Germ-line transmission of the altered GP Ibα allele produced mice with heterozygous GP Ibα alleles. Heterozygous mice were bred to each other to generate a GP Ibα knockout mouse colony. Shown is a representative autoradiograph of mouse DNA digested with HindIII and hybridized with probes flanking the mouse GP Ibα gene. The results provide examples of the wild-type GP Ibα genotype (+/+), a heterozygous GP Ibα locus (+/−), and a homozygous-deficient GP Ibα locus (−/−). (C) Northern analysis of bone marrow isolated from the three representative GP Ibα genotypes is shown. The RNA was electrophoresed through a denaturing 1% agarose/formaldehyde gel and hybridized to probes from the coding region of the mouse GP Ibα gene. A transcript of approximately 2.8 kb is seen in both normal and heterozygous animals, but absent in RNA from GP Ibα knockout mice. The same filter was reprobed with a radiolabeled fragment of the 18S rRNA gene to demonstrate a similar load of RNA in each gel lane.