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Androgenetic haploid embryonic stem cells (AG-haESCs) hold great promise for exploring gene functions and generating gene-edited semi-cloned (SC) mice. However, the high incidence of self-diploidization and low efficiency of SC mouse production are major obstacles preventing widespread use of these cells. Moreover, although SC mice generation could be greatly improved by knocking out the differentially methylated regions of two imprinted genes, 50% of the SC mice did not survive into adulthood. Here, we found that the genome-wide DNA methylation level in AG-haESCs is extremely low. Subsequently, downregulation of both de novo methyltransferase Dnmt3b and other methylation-related genes was determined to be responsible for DNA hypomethylation. We further demonstrated that ectopic expression of Dnmt3b in AG-haESCs could effectively improve DNA methylation level, and the high incidence of self-diploidization could be markedly rescued. More importantly, the developmental potential of SC embryos was improved, and most SC mice could survive into adulthood.

stem cell reports

Reduced Self-Diploidization and Improved Survival of Semi-cloned Mice Produced from Androgenetic Haploid Embryonic Stem Cells through Overexpression of Dnmt3b