FIG. 2.
Disruption of NMDMC in ES cells. (A) Gene-targeting strategy. A partial restriction map of the nmdmc gene is displayed (top). Exons are numbered and indicated by shaded boxes, and thin lines represent introns. The location of probes for hybridization (E2/161, E8/504, and neo) and primers for PCR (P1, P2, and P3) are indicated. The targeting vector pMC1neoΔ3-8/TK is shown, and the predicted targeted allele is indicated. The vector contains a 5′ arm, including exon 2 and part of exon 3, and a 3′ arm including part of exon 8. The E2/161 probe is specific for BamHI fragments containing exon 2 and detects a 5-kb fragment from the wild-type allele and an 11-kb fragment from the targeted allele generated by BamHI digestion. The E8/504 probe is specific for BamHI fragments containing exon 8 and hybridizes to an 8.5-kb fragment from the wild-type allele and an 11-kb fragment from the targeted allele. The neo probe hybridizes to the neomycin cassette. Abbreviations used: B, BamHI; E, EcoRI; H, HindIII; X, XbaI; NEO, neomycin resistance gene; and TK, herpes simplex virus thymidine kinase gene. (B) Southern blot analysis of DNA isolated from ES cell clones. BamHI-digested ES cell DNA was hybridized with E2/161, E8/504, or neo probes. The size (in kilobases) of the NMDMC-specific BamHI fragments is indicated on the left. KO, knockout; wt, wild type.