Targeting of both alleles of the AP-2 gene. (A) Targeting strategy. The wt AP-2 gene (Top) and original neo-targeted disruption (6) (Bottom) are shown along with the pHygKO vector and the recombinant allele derived from this targeting construct (Middle). Exons 5, 6, and 7 are indicated by black boxes; TK, thymidine kinase gene; restriction enzyme sites (R, EcoRI; S, SacI; H, HindIII; P, PstI; B, BglII) and locations of 5′ and 3′ external probes are also indicated. (B) (Left) Southern blot of EcoRI-digested DNA from heterozygous ES cell line (+/−) and AP-2-null line TN63 (−/−) probed with 5′ fragment. In TN63, the remaining 12-kb wt fragment is converted to 7 kb by homologous recombination. (Right) Southern blot of SacI-digested DNA from heterozygous ES cell lines (+/−) and AP-2-null lines TN91 and TN95 (−/−) by using the 3′ probe. The 14-kb neo-disrupted allele is constant; the 7.5-kb wt fragment is converted to 8.5 kb by homologous recombination.