FIG. 5.

mMof is essential for proliferation and/or viability of ES cells. (A) Conditional inactivation of mMof. A partial restriction map of the genomic region containing mMof exons 1 to 4 (black boxes numbered 1 to 4) is shown at the top, followed by a diagram of the targeting vector used for insertion of the FRT-flanked neomycin cassette and loxP sites into the mMof gene (bottom). A diphtheria toxin A gene cassette is also included in the targeting construct as a negative selection marker. The wavy line represents plasmid sequence. The restriction sites shown are BglII (B), EcoRI (E), SalI (S), and SphI (Sp). (B) Southern blot analysis showing cre-mediated recombination of the floxed allele in mMof^flox/+/Rosa26^creERT2/+ and mMof^Δflox/flox/Rosa26^creERT2/+ ES cells after tamoxifen treatment. Genomic DNA was prepared from mMof^Δflox/flox/Rosa26^creERT2/+ (lanes 1, 3, 4, 7, 8, 11, 12, 15, and 16) or mMof^flox/+/Rosa26^creERT2/+ ES cells (lanes 2, 5, 6, 9, 10, 13, 14, 17, and 18) collected at 0, 24, 36, 48, and 60 h from cells treated with 200 nM tamoxifen (Tam) or not treated with tamoxifen (−), digested with PstI, and probed with the 5′ external probe. Note that after 24 h, the conditional (Cond) Mof^flox allele is fully recombined in the presence of tamoxifen. The positions of molecular size markers in lane M (in kilobases) are indicated to the left of the gel. WT, wild type. (C) Western blot analysis for MOF and H4K16ac levels in mMof^Δflox/flox/Rosa26^creERT2/+ ES cells harvested at 0, 24, 48, and 60 h from cells treated with tamoxifen (Tam) (+) or not treated with tamoxifen (−). (D) Proliferation of mMof^flox/+/Rosa26^creERT2/+ and mMof^Δflox/flox/Rosa26^creERT2/+ ES cells with or without tamoxifen treatment. Cell numbers were determined at different time points to determine the population doublings in cells with and without mMof.