FIG. 1.

Targeted mutation of mdc9. (A) The genomic structure of the wild-type mdc9 allele is shown at the top. The position of the 5" probe used for Southern blot analysis is indicated, as well as the targeted exon, EcoRI (E) sites, and ApaI and NcoI sites. The targeting vector is shown in the middle. The targeted exon was disrupted by insertion of a pMC1neoPolyA cassette, which introduces an additional NcoI site. A diphtheria toxin (Diphth. toxin) gene cassette was included for negative selection. The bottom panel shows the genomic structure of the targeted allele. The additional NcoI site introduced into the targeted allele by homologous recombination reduces the size of the NcoI-digested genomic fragment that is recognized by the 5" probe from ∼20 to ∼11 kb. (B) Southern blot analysis of NcoI-digested genomic DNA from wild-type, heterozygous mdc9^+/−, and homozygous mdc9^−/− mice. (C) Western blot analysis of MDC9 expression in mouse lung (lanes 1 and 2), brain (lanes 3 and 4) or embryonic fibroblasts (MEF; lanes 9 and 10) from wild-type or mdc9^−/− mice confirms the lack of MDC9 expression in mdc9^−/− mice. Lanes 1 to 4 were probed with a polyclonal antiserum (pAb) against the MDC9 cytoplasmic tail. Lanes 9 and 10 were probed with a mouse monoclonal antibody (mAb) against mouse MDC9 which was generated from mdc9^−/− mice immunized with an IgG-Fc fusion protein with the ectodomain of MDC9 (see Materials and Methods). This monoclonal antibody recognizes only nonreduced MDC9. Identical samples to those in lanes 1 to 4 were probed with a polyclonal antiserum against the cytoplasmic domain of MDC15 (lanes 5 to 8).