FIGURE 1.

Functional disruption of α_D by homologous recombination. A, The murine α_D genomic locus containing exons 1–10 is represented (WT). Genomic clones were isolated from a 129Sv mouse genomic λ DNA library using oligonucleotide probes specific to the α_D I domain. The largest (clone 3.1) was 14 kb in length and contained exons 1–6 as determined by Southern blot analysis and nucleic acid sequencing. A second 10-kb genomic clone (4.1) that overlapped with the 3′ end of 3.1 and contained exons 6–10 was also isolated. A 7.5-kb genomic fragment containing exons 1–5 and a 5′ region of exon 6 were cloned from 3.1 and inserted upstream of three in-frame stop codons in a polylinker modified pBlueScript vector. A 3-kb Xmn1-BglII fragment containing the 3′ end of exons 6 and 7 was cloned downstream of this fragment to generate a novel α_D allele leading to termination of translation in the I domain following residue 153(Ile). The novel α_D allele was cloned between two herpes simplex TK genes and a floxed neomycin selection cassette was inserted 3′ to the stop codons in exon 6 to generate the targeting vector. The targeting vector was linearized with XhoI and electroporated into TC1 embryonic stem cells. Recombinant embryonic stem cells were selected in the presence of G418 and FIAU as previously described (18) and were identified by Southern blot analysis using BglII and a 5′ flanking BamHi/BgIII probe located outside the targeted region. Positive cell lines were confirmed using XmnI digestion and hybridization to a probe corresponding to the neomycin gene. A single recombinant cell line (1e-1) was used for blastocyst injection to create recombinant mice. The floxed neomycin selection cassette was removed by mating to a transgenic mouse line that expresses Cre recombinase (19). Mice containing α_D^loxP and WT alleles were genotyped by PCR using isolated tail DNA. The forward (5′-GGACCCCAGGACACAGTTGAG-3′) and reverse (5′-CACAGGCCACAGTGTACAGTATT-3′) primers amplify a 268-bp band including the inserted loxP site from the recombinant allele and 215 bp from the WT allele. The recombinant mouse line was subsequently backcrossed and maintained on a C57BL/6 background. B, Southern blot analysis of genomic DNA isolated from WT embryonic stem cells and the α_D-recombinant embryonic stem cell line (1e-1). Genomic DNA digested with BglII was probed with a 600-bp BamH1-BglII fragment from the α_D genomic clone. This 5′ probe hybridized to both the 11-kb fragment derived from the WT allele and the 8-kb fragment corresponding to the recombinant allele. The genomic structure of the 3′ end of the α_D^neo allele was confirmed in the 1e-1 clone by hybridization of the targeting vector-specific neomycin probe to the 6.5-kb XmnI genomic fragment.