Effects Of DNA Methylation At The Imprinting Control Region Of H19 Gene On Mouse Parthenogenetic Embryonic Stem Cells And Parthenogenetic Embryos

The failure of mammalian parthenogenetic embryos developed to term is associated with the abnormal expression of imprinted genes,in which DNA methylation plays an important role.In this study,the expression and the DNA methylation of imprinted genes were detected in isolated mouse parthenogenetic embryonic stem cells.Then,CRISPR/d Cas9-Dnmt3a vector which was used to target the imprinting control region of H19 gene,the effects of DNA methylation on the development of mouse parthenogenetic embryonic stem cells and parthenogenetic embryos were investigated.1.Establishment and identification of mouse parthenogenetic embryonic stem cells(PESCs)After activated by SrCl₂,the parthenogenetic embryos at blastocyst stage were cultured in N2B27 medium,and the parthenogenetic embryonic stem cell lines PESCs were obtained after subcultured.The morphology of PESCs was similar to that of normal mouse embryonic stem cell(ESCs),eighty-one percent of cells have normal number of chromosomes(2n=40);PESCs can form embryoid bodies with three dermal differentiation in vitro,but fail to form teratoma in vivo.In PESCs,the expression of maternal imprinted gene Igf2 was lower than that in ESCs,the promoter region of the maternal imprinted gene Snrpn was hypermethylated;The expression of the paternal imprinted gene H19 was high,and the promoter region of H19 was hypomethylated.2.Function of DNA methylation in the imprinting control region(ICR)of H19GeneDNA methylation status in the imprinting control region affects the expression regulation of H19/Igf2.The results showed that among the four CTCF binding sites,DNA methylation of CTCF4 binding sites were significantly lower in PESCs than that in ESCs.To investigate the regulation of Igf2 expression by DNA methylation modification in H19 ICR in PESCs,the CRISPR/d Cas9-Dnmt3a vector was constructed,and the CTCF4-sg RNA was designed to target the binding site of CTCF4;After digestion by Bam HI and Xho I,CTCF4-sg RNA was ligated to the empty pg RNA-humanized vector,the construction of the sg RNA-humanized vector was verified by sequencing.The CRISPR/d Cas9-Dnmt3a vector and sg RNA-humanized vector were co-transfected into PESCs by lentivirus transfection,the DNA methylation of the CTCF4 binding site at H19 ICR was significantly increased,the expression of Igf2m RNA was increased and H19 m RNA was decreased.Ch IP assay showed that the binding of CTCF protein to the H19 ICR was reduced after transfection,while the expression of CTCF had no significant difference.In order to investigate the effects of DNA methylation modification in H19 ICR on embryos development,the activated parthenogenetic embryos were micro-injected with the targeting vectors,after embryo transfer,a malformed embryos were observed by dissection,no normal mouse is delived.In summary,CRISPR/d Cas9-Dnmt3a can be used to modify the DNA methylation status of the CTCF binding sites at H19/Igf2 gene locus,which affect mouse parthenogenetic embryonic stem cells and the development of parthenogenetic embryos by regulating gene expression.These results provide a basis for clarifying the regulation of the imprinting gene cluster of H19/Igf2 and for further investigating the development mechanism of parthenogenetic embryos.