FIG. 5.

Dnmt3b6 has no enzymatic activity in vivo. (A) Strategy of targeted deletion of Dnmt3b exons 21 and 22. The top line shows the Dnmt3b genomic structure with exons represented by vertical bars. The targeting vector (second line) was constructed by replacing exons 21 and 22 with a PGK-puromycin cassette. A PGK-DTA cassette was introduced for negative selection to increase the targeting frequency. (B) Southern analysis of the genotype of ES cell lines. Genomic DNA was digested with EcoRV and hybridized to a 3′ external probe, as shown in (A). The 16-kb wild-type allele, the 5-kb Dnmt3b1 targeted allele, and the 14-kb Dnmt3b null allele (30) are indicated. (C) Lysates from the indicated cell lines were immunoblotted with anti-Dnmt3b (top), anti-Dnmt3a (middle), and anti-tubulin (bottom) antibodies. (D and E) Genomic DNA from the indicated ES cell lines was digested with HpaII and hybridized to probes for endogenous C-type retrovirus repeats (D) and minor satellite repeats (E).