小鼠卵细胞质对供体核DNA甲基化和U2afbp-rs基因表达的调控  

Regulation of DNA Methylation and U2afbp-rs Gene Expression in Early pre-implantation Cloned Mice

作　　者：李少华[1] 于建宁[1] 王丹秋[1] 武玮[1] 林飞[1] 刘红林[1] 

机构地区：[1]南京农业大学动物科技学院,南京210095

出　　处：《生物化学与生物物理进展》2007年第12期1260-1268,共9页Progress In Biochemistry and Biophysics

基　　金：国家自然科学基金资助项目(30471244)~~

摘　　要：利用细胞核移植技术将NIH3T3细胞核和孤雌桑椹胚单个卵裂球,分别移植到去核MⅡ期受体卵母细胞中,通过免疫荧光染色后比较体外受精胚、孤雌胚、NIH3T3核移植重组胚和孤雌桑椹胚核移植重组胚附植前各时期胚胎DNA甲基化水平的变化,以探明克隆胚细胞核去分化与DNA甲基化的相互关系.利用Real-timePCR技术检测体外受精胚、孤雌胚和孤雌桑椹胚核移植重组胚附植前各时期胚胎中,印记基因U2afbp-rs基因以及非印记基因eIF-4C基因表达量的变化,以探明小鼠卵细胞质对克隆胚细胞核中印记基因表达的调控.结果表明,克隆胚供体核基因组DNA在核移植后并没有发生主动去甲基化.孤雌桑椹胚核移植后重组胚中U2afbp-rs基因和eIF-4C基因的表达水平要显著低于对照孤雌胚,但其表达量变化规律与对照孤雌胚相同,说明了卵细胞质对供体核印记基因的表达具有一定的调控作用.Embryos that were produced by parthenogenesis were cultured to morula, then each cell of morulas was transferred into enucleate M Ⅱ oocytes, as called parthenogenetic morula nuclear transplantation embryos here. NIH3T3 cell nucleus were also transferred into enucleate M Ⅱ oocytes. In order to prove up the relationship between nuclear reprogram and DNA methylation during the process of nuclear transplantation, the two cloned pre-implantation embryos together with in vitro fertilization （IVF） and parthenogenetic embryos were subjected to immunocytochemistry with antibody to DNA methylation （5＇-MeC）. In order to research the regulation of oocyte cytoplasm to the relative expression level of imprint genes, the relative expression level of U2αfbp-rs gene which is maternally imprint and non-imprint elF-4C gene in pre-implantation IVF, parthenogenetic and parthenogenetic morula nuclear transplantation embryos by real-time PCR were detected.The results show that neither the genomic DNA of NIH3T3 cloned embryos nor parthenogenetic morula nuclear transplantation embryos were demethylated actively. Although both the relative expression level of U2αfbp-rs gene and elF-4C gene in parthenogenetic morula nuclear transplantation embryos were lower than control parthenogenetic embryos, the expression of the two genes were similar to the control parthenogenesis group. These results suggest that the imprinting gene expression of donor nuclear are regulated by oocyte cytoplasm.

关 键 词：核移植 DNA甲基化 基因组印记 基因表达调控 核重序 U2αfbp-rs 

分 类 号：S813[农业科学—畜牧学]