Tet3蛋白在山羊早期胚胎DNA甲基化重编程过程中的作用

项目名称： Tet3蛋白在山羊早期胚胎DNA甲基化重编程过程中的作用

项目编号： No.31302045

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： 刘军

作者单位： 西北农林科技大学

项目金额： 25万元

中文摘要： DNA去甲基化是胚胎发育和细胞重编程过程中的重要环节，Tet3蛋白作为小鼠受精卵雄原核主动去甲基化的关键酶，是胚胎发育所必须的蛋白因子。山羊早期胚胎去甲基化模式与小鼠不同，去甲基化机制尚不清楚，而且克隆胚胎普遍存在DNA去甲基化不完全现象。因此，本课题拟研究Tet3蛋白在山羊早期胚胎DNA主动去甲基化过程中的作用，并探究Tet3蛋白能否提高克隆胚胎DNA甲基化重编程效率。项目拟通过RNA干扰技术下调卵母细胞中Tet3蛋白表达，显微受精后检测胚胎全基因组和特定位点5mC和5hmC的水平；利用诱导型表达载体控制Tet3蛋白在体细胞中表达，核移植后检测克隆胚胎全基因组和特定位点5mC和5hmC的水平，并检测相关基因的表达，从而确定Tet3蛋白对山羊早期胚胎DNA甲基化重编程的作用。本课题的实施对揭示山羊早期胚胎DNA甲基化重编程机制，提高动物克隆效率有重要意义。

中文关键词： 体细胞核移植；DNA 甲基化；Tet 蛋白；重编程；胚胎发育

英文摘要： DNA demethylation is a crucial step during embryonic development and somatic cell reprogramming processes. Ten-eleven translocation (Tet3) protein, a key enzyme for active demethylation of paternal pronucleus in mouse zygote, is essential to activate the expression of pluripotency genes for maintaining the embryonic development. The mechanism of DNA demethylation in goat embryos is defferent from in mouse, and it is still unclear. In addition, incomplete DNA demethylation prevalently takes place in the cloned embryos. The purpose of the project is to investigate the effects of Tet3 protein on the active demethylation of goat early embryo development, and explore whether the Tet3 protein can improve the DNA methylation reprogramming process of cloned embryos. The expression level of Tet3 will be silenced by RNA interfering technique, and the levels of 5mC and 5hmC in genome-wild and specific loci in genome will be eximined after intracytoplasmic sperm injection. Tet3 protein will be overexpressed in somatic cells by transfection with an inducible expression vector. The levels of 5mC and 5hmC in genome-wild and specific loci in genome and the expression level of pluripotency and imprinring genes of cloned embryos will be eximined after nuclear transfer. The findings ot this proposal will demonstrate the the roles

英文关键词： Somatic cell nuclear transfer；DNA methylation；Tet protein；Reprogramming；Embryonic development