Tet3调节的DNA去甲基化对牛体细胞核移植胚胎早期发育的影响和机制研究

项目名称： Tet3调节的DNA去甲基化对牛体细胞核移植胚胎早期发育的影响和机制研究

项目编号： No.31472093

项目类型： 面上项目

立项/批准年度： 2015

项目学科： 畜牧学与草地科学

项目作者： 唐博

作者单位： 吉林大学

项目金额： 82万元

中文摘要： 细胞核不完全重编程导致动物克隆效率低下、后代发育异常，严重制约体细胞核移植（SCNT）技术的发展和应用。而DNA去甲基化异常是不完全表观重编程的重要原因之一。本实验室前期研究发现，牛合子中Tet3催化早期胚胎发生DNA去甲基化。以此为基础，本项目拟运用免疫荧光染色、亚硫酸盐测序、siRNA等技术，以不同发育阶段的体外受精和SCNT胚胎（合子、4-细胞、囊胚）为研究对象，检测全基因组5mc/5hmc水平、全能性相关基因和印记基因的表达及启动子区的甲基化状态，并评估囊胚质量，从而明确各阶段的SCNT胚胎DNA去甲基化动态变化；以及Tet3是否直接参与SCNT胚胎的去甲基化过程；旨在发现Tet3及其催化的DNA去甲基化对SCNT胚胎发育的影响，并初步阐明此影响的作用机制。进而揭示DNA去甲基化和SCNT早期胚胎发育的相关性，为提高牛克隆效率、开展转基因克隆牛育种工作等生产实践提供丰富的理论依据。

中文关键词： DNA去甲基化；Tet3基因；体细胞核移植；DNA甲基化；体外受精

英文摘要： The incomplete reprogramming of nucleus resulting in the inefficiency of animal cloning and abnormal development of offspring, seriously constraints the application and development of somatic cell nuclear transfer (SCNT). One important reason for the incomplete epigenetic reprogramming is the disorder of DNA demethylation. Our previous research found Tet3 is the main enzyme to catalyze the DNA demethylate during the bovine zygote period. Therefore, through immunofluorescence staining, bisulfite sequencing and siRNA technology, we research the genome-wide 5mC/5hmC levels, the methylation situation of promoter region and expression dynamic changes of pluripotency-related genes and imprinted genes at the three different stage of IVF and SCNT embryo development (zygotes, 4 cell stage and blastocyst stage), and evaluate the quality of SCNT embryos. Then we can uncover the DNA methylation levels dynamic changes at the different embryos development stages, and whether Tet3 directly involved in the DNA demethylation of SCNT embryos. Thus, we can clarify the mechanism of the influence of Tet3 and Tet3 catalyzed DNA demethylation on the development of SCNT embryos. This will provide more experiment basis to the relevance of DNA demthylation and SCNT early embryos development, and provide rich theoretical basis for improving the efficiency of bovine SCNT works and transgenic cloned cattle breeding works.

英文关键词： DNA demethylation;Tet3 gene;SCNT;DNA methylation;IVF