FIG. 5.

Deficiency in either Dnmt3a or Dnmt3b reduces DNA methylation at the Oct4 promoter in ES cells and embryos. (A) Confirmation of wild-type (wt) and knockout ES cell lines by Western blotting. (B) COBRA methylation analysis of the Oct4 promoter. Wild-type and knockout ES cells were induced with RA for up to 7 days, and the methylation levels of Oct4 at different time points were assessed by TaqI restriction assay of the PCR products derived from bisulfite-treated genomic DNA and quantified (bottom). Labeling of the digested fragments is as in Fig. 4C. (C) Bisulfite sequencing analysis of Oct4 and Nanog at day 3 upon RA treatment. Percentages of the methylated CpG sites (filled circles) are indicated. (D) Bisulfite sequencing analysis of Oct4 in mouse embryos. Genomic DNA was from wild-type (wt), single knockout (3aKO and 3bKO), or double knockout (DKO) embryos collected at E9.5 (three for each genotype).