Figure 2.

Characterization of the null allele. (A1) A 12-kb EcoRI fragment from the genomic clone was subcloned into pBSIISK⁺. A 1-kb BglII to XhoI fragment (F) was removed. This fragment was subcloned and used as an external probe for Southern blot analysis. (A2) A fragment of DNA containing the neomycin resistance gene (NEO^r; ref. 27) was inserted between the BamHI and BglII sites that flank exon 6, deleting exon 6 and portions of the flanking introns. The resulting genomic fragment was purified and ligated into the vector containing two copies of the herpes simplex virus thymidine kinase gene (27). The final construct pTKNEOURO-D was linearized with XhoI and transferred by electroporation into ES cells. (B) A restriction map of the wt allele (1) and the NEO^r disrupted allele (2) are shown. Approximate band size expected from digestion with EcoRI are shown as solid lines below each allele. Horizontal arrows represent primers (WF, wt forward; R, reverse; NF, NEO forward) used to generate PCR products shown in D. (C) Southern blot of wt DNA and DNA from ES cell lines carrying the targeted disruption. The DNA was digested to completion with EcoRI and probed with a BamHI, EcoRI fragment of the F probe or a portion of the NEO gene. Molecular size markers are indicated on the right. (D) Genotyping by PCR. Genomic DNA was prepared from tails of mice and used for genotypic assignment (27). The three primers shown in B1 and B2 were used to amplify genomic DNA. The bands at 196 bp and 298 bp represent the disrupted (Null) and wt alleles, respectively. Restriction enzymes: BamHI (B), BglII (Bg), EcoRI (E), HindIII (H), NotI (N), XhoI (X), and XbaI (Xb).