Fig. 2. Targeted disruption of Dnmt3L and analysis of DNA methylation in Dnmt3L^–/– mutant ES cells. (A) Gene targeting at the Dnmt3L locus. The top line shows the wild-type genomic locus. The vertical bars represent the exons starting from the first coding exon 1 to exon 11. The targeting vectors were constructed by replacing exons 2-7, which encode the entire PHD domain and part of motif 1, either with an IRES-ßgeo or a PGK-hygromycin (Hyg) cassette. S, SacII; N, NsiI; RV, EcoRV; and B, BamHI. (B) Southern analysis of the genotype of mutant ES cell lines. Genomic DNA was digested with NsiI and hybridized with a 3' external probe. J1, the wild-type parental line; 277 and 280, Dnmt3L^+/– lines; and 196, a Dnmt3L^–/– line (ßgeo/hyg). (C-E) Genomic DNA from wild-type and mutant ES cell lines, as described in B, was digested with designated enzymes and hybridized to the MMLV, Igf2 DMR2, and the Igf2r region 2 probes. Two independent Dnmt3L^–/– lines, 80 and 196, were used. No significant difference in DNA methylation was detected between wild-type and Dnmt3L^–/– lines. m, maternal allele: p, paternal allele.