Genetic evidence for Dnmt3a-dependent imprinting during oocyte growth obtained by conditional knockout with Zp3-Cre and complete exclusion of Dnmt3b by chimera formation.

In the male and female germ-lines of mice, both of the two de novo DNA methyltransferases Dnmt3a and Dnmt3b are expressed. By the conditional knockout experiments using the Tnap-Cre gene, we previously showed that deletion of Dnmt3a in primordial germ cells disrupts paternal and maternal imprinting, however, Dnmt3b mutants did not show any defect. Here, we have knocked out Dnmt3a after birth in growing oocytes by using the Zp3-Cre gene and obtained genetic evidence that de novo methylation by Dnmt3a during the oocyte growth stage is indispensable for maternal imprinting. We also carried out DNA methylation analysis in the mutant oocytes and embryos and found that hypomethylation of imprinted genes in Dnmt3a-deficient oocytes was directly inherited to the embryos, but repetitive elements were re-methylated during development. Furthermore, we show that Dnmt3b-deficient cells can contribute to the male and female germ-lines in chimeric mice and can produce normal progeny, establishing that Dnmt3b is dispensable for mouse gametogenesis and imprinting. Finally, Dnmt3-related protein Dnmt3L is not only essential for methylation of imprinted genes but also enhances de novo methylation of repetitive elements in growing oocytes.