ZFP57与牛克隆胚印迹基因异常去甲基化的关系研究

项目名称： ZFP57与牛克隆胚印迹基因异常去甲基化的关系研究

项目编号： No.31302046

项目类型： 青年科学基金项目

立项/批准年度： 2014

项目学科： 农业科学

项目作者： 苏建民

作者单位： 西北农林科技大学

项目金额： 25万元

中文摘要： 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。

中文关键词： 克隆胚胎；印迹基因；ZFP57；DNA 甲基化；

英文摘要： 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.

英文关键词： Cloned embryo；Imprinted gene；ZFP57；DNA methylation；