Fig. 7. Methylation of imprinted genes in embryos derived from a transplanted [Dnmt3a^–/–, Dnmt3b^+/–] ovary. (A-D) Methylation of four imprinted genes (Igf2r, Peg1, Peg3, and Snrpn) was analyzed either by Southern blot hybridization (A,B) or bisulfite sequencing (C,D) with DNA isolated from E9.5 wild-type (+/+), Dnmt3a^+/– (+/–), and Dnmt3a^–/– (–/–) embryos, and two embryos derived from the [Dnmt3a^–/–, Dnmt3b^+/–] oocytes (labeled as 1, 2). In A and B, DNA was digested with designated restriction enzymes and Southern blots were hybridized with probes of Igf2r region 2 (A) or Peg1 DMR (B). Bisulfite sequencing analysis of methylation (black circles) of Peg3 DMR and Snrpn DMR1 in wild type and embryos 1 and 2 derived from ovary transplantation (OVT) (C,D). The maternal alleles of Igf2r, Peg1, Peg3 and Snrpn were almost completely unmethylated in the two embryos derived from the [Dnmt3a^–/–, Dnmt3b^+/–] mother, but they were methylated in Dnmt3a^+/– and Dnmt3a^–/– embryos from Dnmt3a^+/– mothers. (E,F) Methylation of H19 (E) and MMLV (F) remained unchanged. Methylation of the paternally imprinted H19 gene and non-imprinted endogenous MMLV DNA was unaffected in all embryos tested.