Generation of chimeric mice and progeny genotyping.

(A) Schematic representation of the wild-type α_IIballele, the targeting vector, and the resultant recombinant α_IIb locus. Filled rectangles indicate exons. Arrows indicate putative transcription initiation sites. Restriction endonuclease enzymes are abbreviated as follows: H,HindIII; B, BamHI; E, EcoRI; Hc, HincII; RV, EcoRV. Probe A was produced by PCR amplification using oligonucleotides 5′-GCCCATGTGTGTGTATGCC-3′ and 5′-CCTCAGGTCTCCATCTTGAACC-3′, located within the 5′-flanking region of the α_IIb gene. Probe B was produced using 2 oligonucleotides, 5′-ACACAGAGAGACCCCCTGTC-3′ and 5′-CGGTAGCTGGAGATGATGTTC-3′, located within intron 6 and exon 7, respectively. Both probes were labeled by a random priming method using fluorescein-11-dUTP and hybridized according to the manufacturers' recommendations (Gene Image Random prime labeling and CDP star detection System, Amersham). (B) Southern blot analysis ofHindIII and EcoRV double-digested DNA of R1 ES cell line (WT) and recombinant clones (lanes 1-4) hybridized with probe A. Targeted clones have both the wild-type (6.3 kb) and the disrupted (3 kb) alleles. (C) Southern blot analysis of HincII andBamHI double-digested DNA of R1 ES cell line (WT) and recombinant clones (lanes 1-4) hybridized with probe B. Recombinant clones have wild-type (4.3 kb) and mutated (7.6 kb) alleles. (D) Southern blot analysis of progeny derived from heterozygous intercross. Tail DNA digested with HindIII and subsequently hybridized to probe A indicated the presence of all expected genotypes, including the predicted 6.3-kb band for the wild-type allele and the 5-kb fragment for the mutant allele. A wild-type (+/+), a heterozygous (+/−), and a homozygous mutant (−/−) offspring are represented.