印记基因Xist及H19对猪体细胞核移植重编程的影响及修复研究

Somatic cell nuclear transfer (SCNT) technology plays an irreplaceable role in the research of embryonic development, cells epigenetic reprogramming and transgenic animals. However, there are many problems need to be solved, especially the low efficiency. Previous study on the cloned mice revealed that many imprinted genes abnormally expressed and seriously affected the development of cloned embryos. Gene targeting of Xist and H19 gene greatly improved the efficiency of cloning mice. Our preliminary study on the cloned pigs also found that Xist and H19 gene were abnormally high expressed, but the expression of IGF2 associated with the embryo development was significantly reduced. So in this project, we are aimed to study the genomic imprinting during reprogramming of porcine SCNT. Firstly the expression of Xist, H19 and related genes will be detected in SCNT embryos with various developmental stages. Using the most advanced Next-generation- sequencing technology, the analysis of gene expression profiling and genome-wide methylation of the SCNT embryos will be performed to study the effects of imprinted genes on porcine SCNT reprogramming. On this basis, the most high-fidelity eSpCas9 or SpCas9-HF1 will be applied to target porcine endogenous Xist and H19 gene in somatic cells, attempt to repair the abnormal genetic imprinting, and improve the efficiency of porcine SCNT.

体细胞核移植技术在胚胎发育、体细胞表观遗传学重编程及转基因动物研究中具有不可替代的作用，但其效率低的问题一直未得到解决。前期对克隆小鼠的研究发现克隆胚胎中许多印记基因异常表达，严重影响克隆胚胎的发育。利用基因打靶技术，靶向修饰Xist和H19基因极大的提高了克隆小鼠的效率。我们在前期克隆猪的研究中发现猪克隆胚胎Xist和H19基因异常高表达而与发育相关的IGF2表达显著下调。因此本项目拟利用体细胞核移植获得早期不同发育阶段的胚胎，首先检测Xist和H19及其相关基因在各阶段的表达差异；继而利用目前最先进的二代测序技术深入分析猪克隆胚胎的基因表达谱及全基因组甲基化水平，着重研究印记基因对猪体细胞重编编程的影响；在以上基础上利用目前最新的高保真eSpCas9或SpCas9-HF1在体细胞水平靶向修饰猪内源Xist和H19基因，尝试修复核移植重编程引起的印记异常，以提高体细胞核移植效率。

通过体细胞核移植（SCNT）技术获得转基因猪不仅在农业育种而且在生物医药领域中均具有重要作用,然而过低的克隆效率严重影响了基因修饰克隆猪的应用。本研究中我们首次系统地比较了克隆胚胎和受精胚胎植入前后的转录组差别。我们发现，克隆异常的胚胎出现全染色体水平的基因下调，包括X染色体和每条常染色体。许多基因，如负责调控X染色体沉默的基因XIST，在猪异常克隆胚胎中显著地高表达。利用TALEN技术，敲除供体细胞中XIST，可以拯救每条染色体上原本失活或者下调的基因，从而显著提高猪克隆胚胎的发育潜力。我们进一步发现，XIST敲除的胚胎，其发育潜力的提高与全染色体水平的组蛋白H3K9me3的下调相关。通过向猪克隆胚胎注射H3K9me3的特异去甲基化酶Kdm4A可以显著地降低整体H3K9me3的水平。但是，Kdm4A的注射同时诱导了XIST的异常高表达，原因在于体细胞中XIST启动子区是H3K9me3高度富集的。注射Kdm4A从而降低了克隆胚胎的发育潜能。将Kdm4A注射后的胚胎移植到代孕母体，并未提高猪克隆效率。将XIST基因敲除的胚胎移植到代孕母体，相对于对照组胚胎，克隆效率提高了约6.9倍。本项目利用基因靶向修饰技术修复体细胞核移植胚胎基因印记异常、提高猪克隆效率的研究提供了可靠的理论依据。