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-neor cassette (neor). 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.