采用dCas9-SunTag-DNMT3A技术调控玻璃化冷冻牛卵母细胞IVF囊胚中IGF2R基因甲基化水平

The Regulation of Methylation Level of IGF2R Gene in IVF Blastocysts Derived from Vitrified Bovine Oocytes by dCas9-SunTag-DNMT3A Technology

旨在探究dCas9-SunTag-DNMT3A编辑系统对玻璃化冷冻牛卵母细胞IVF囊胚中IGF2R基因甲基化水平及胚胎发育能力的影响,为冷冻卵母细胞/胚胎特定位点DNA甲基化的精确调控奠定基础。本研究将经过体外成熟的牛卵母细胞进行玻璃化冷冻,随后进行体外受精,将受精所得到的原核胚进行dCas9-SunTag-DNMT3A编辑系统的注射,统计并计算卵母细胞的发育情况;通过亚硫酸盐测序的方式检测IGF2R基因启动子的甲基化水平,并利用荧光定量PCR检测IGF2R及相关基因的表达水平。与冷冻组相比,注射不同浓度的dCas9-SunTag-DNMT3A编辑系统后,只有40 ng·μL^(-1)组显著地提高了玻璃化冷冻卵母细胞IVF后的发育能力(P<0.05),20和60 ng·μL^(-1)组间差异不显著(P>0.05),但40 ng·μL^(-1)组发育效果仍然显著低于新鲜对照组(P<0.05);对检测冷冻组、新鲜组、40 ng·μL^(-1)组IGF2R基因启动子甲基化水平分析发现,40 ng·μL^(-1)组水平与新鲜组相似,显著高于冷冻组(P<0.05);荧光定量试验结果显示,40 ng·μL^(-1)组IGF2R基因mRNA表达水平相较于冷冻组显著降低(P<0.05),与新鲜组相似。注射40 ng·μL^(-1)的dCas9-SunTag-DNMT3A甲基化编辑系统能够通过有效升高IGF2R基因启动子甲基化水平(P<0.05)及显著降低其mRNA表达水平(P<0.05),来正向调节玻璃化冷冻卵母细胞IVF胚胎的发育情况,提高胚胎发育能力,使得其卵裂率和囊胚率都得到显著提高(P<0.05),同时促进胚胎发育相关基因的表达。

The study aimed to explore the effect of dCas9-SunTag-DNMT3A editing system on the methylation level of IGF2R gene and embryonic developmental ability in IVF blastocysts from vitrified bovine oocytes,and to lay the foundation for precise control of DNA methylation at specific sites of vitrified oocytes/embryos.In this study,the in vitro matured bovine oocytes were vitrified,and followed by in vitro fertilization,and the prokaryotic embryos obtained by fertilization were injected with dCas9-SunTag-DNMT3A editing system,and the developmental ability of oocytes were analyzed;the methylation level of the IGF2R gene promoter was detected by sulfite sequencing,and the expression levels of IGF2R and related genes were detected by fluorescence quantitative PCR.Compared with the vitrified group,after injection of different concentrations of dCas9-SunTag-DNMT3A editing system,only the 40 ng·μL^(-1) group significantly improved the developmental ability of vitrified oocytes after IVF(P<0.05).There was no significant difference between the 20 and the 60 ng·μL^(-1) groups(P>0.05),but the developmental effect of the 40 ng·μL^(-1) group was still significantly lower than that of the fresh control group(P<0.05).The methylation level of IGF2R gene promoter in the 40 ng·μL^(-1) group was found to be similar to that of the fresh group and significantly higher than that of the vitrified group(P<0.05).The mRNA level of IGF2R gene in the 40 ng·μL^(-1) group was significantly lower than that in the vitrified group(P<0.05),which was similar to the fresh group.Injection of 40 ng·μL^(-1) dCas9-SunTag-DNMT3A methylation editing system can effectively increase the methylation level of IGF2R gene promoter(P<0.05)and significantly reduce its mRNA expression level(P<0.05),to positively regulate the development of IVF embryos derived from vitrified oocyte,so that the cleavage rate and blastocyst rate were significantly increased(P<0.05),and the expression of embryonic development-related genes was promoted.