ZFP57与牛克隆胚印迹基因异常去甲基化的关系研究

The developmental abnormity and low success rate of somatic cell nuclear transfer (SCNT) in mammalian cloning is mostly due to aberrant demethylation of imprinted genes of cloned embryos during nuclear reprogramming. However, little is known about the mechanisms of aberrant demethylation of imprinted genes during SCNT. We recently found that ZFP57, which maintains methylation imprints during early embryonic development, was significantly down-expressed in bovine SCNT embryos compared with the control of IVF embryos from global gene expression analysis , and ZFP57-binding sequences (TGCCGC) were found in all imprinting control region (ICR) of imprinted genes which were hypomethylated in SCNT embryos and calves. Those suggest that ZFP57 may cause aberrant demethylation of imprinted genes of cloned embryos. In order to validate this hypothesis, shRNA plasmid targeting ZFP57 and ZFP57 expression vectors will be constructed and transgenic cells will be obtained. After SCNT with ZFP57 knockdown/expression cells, DNA methylation levels in the 6 ICRs from imprinted genes will be analyzed in preimplantation embryos, to investigate whether aberrant demethylation of imprinted genes of cloned embryos caused by ZFP57, and to investigate whether ZFP57 induced-expression could maintains methylation imprints of cloned embryos. This research may provide new insights into revealing the mechanisms of aberrant demethylation of imprinted genes during SNCT and the cause of low cloning efficiency.

体细胞克隆胚胎重编程过程中普遍存在的印迹基因异常去甲基化是引起克隆动物发育异常和体细胞克隆效率低下的重要原因。但异常去甲基化机理尚不明确。申请人前期研究中发现具有维持印迹基因甲基化功能的ZFP57基因在牛克隆胚胎上异常低表达；并且申请人在牛克隆胚胎及死亡克隆牛犊上发现的异常去甲基化的印迹调控区含有ZFP57的特异识别位点。因而推测ZFP57可能与克隆胚胎上印迹基因异常去甲基化有关。为了验证这一假说，本项目将首先构建ZFP57干扰载体和表达载体并获得转基因细胞株，核移植后在附植前各期胚胎上检测6个印迹调控区的甲基化水平，来分析ZFP57是否与克隆胚胎上印迹基因异常去甲基化有关，并分析通过诱导ZFP57表达是否可以克服上述甲基化丢失的问题。本项目将为阐明体细胞克隆胚胎重编程过程中甲基化印迹异常的机理以及体细胞克隆效率低下的原因提供理论依据。

体细胞克隆胚胎重编程过程中普遍存在的印迹基因异常去甲基化是引起克隆动物发育异常和体细胞克隆效率低下的重要原因。为研究ZFP57在牛早期胚胎发育过程中的作用，申请人设计并合成了ZFP57 mRNA siRNAs，注射到体外受精的受精卵。另外构建ZFP57表达载体并获得转基因细胞株，核移植后在附植前检测各组胚胎的卵裂率和囊胚发育率。并通过分析第七天囊胚总细胞数、滋养层细胞数、内细胞团细胞数以及凋亡细胞数来评估各组胚胎的质量。结果发现干扰ZFP57会影响牛体外受精胚胎发育。诱导表达ZFP57可显著促进牛体细胞克隆胚胎的发育，第七天囊胚发育率高于克隆对照组。诱导表达ZFP57可提高克隆囊胚细胞数、滋养层细胞数、内细胞团细胞数以及滋养层细胞数/内细胞团细胞数比值，另外，诱导表达ZFP57可显著降低克隆囊胚的细胞凋亡率。分析了印迹基因XIST、H19/IGF2和IGF2R的ICR区在各组胚胎上的甲基化水平。发现牛体细胞克隆胚胎上存在严重的印迹甲基化的丢失，ZFP57可纠正牛克隆胚胎上异常的印迹基因低甲基化，使其甲基化水平到达和体外受精胚胎接近的水平。总之，ZFP57可保护牛克隆胚胎发育过程中的印迹基因甲基化，从而提高了牛克隆胚胎的胚胎质量，促进了牛克隆胚胎的发育。本项目将为阐明体细胞克隆胚胎重编程过程中甲基化印迹异常的机理以及体细胞克隆效率低下的原因提供理论依据。