克隆牛重编程的表观遗传机理研究

The application of somatic cell nuclear transfer (SCNT) technology to cloned cattle not only has important economic value but also has important significance for elucidating the reprogramming mechanism in early embryos. We have systematically compared and studied on the expression and epigenetic level, from the autologous cloned embryos (somatic donor cells and the recipient oocyte from the same individual), with those of allogeneic cloned embryos (somatic donor cells and the recipient oocytes from individuals with similar mitochondrial subtypes) or heterogeneous cloned embryos cloned embryos (somatic donor cells and the recipient oocytes from individuals with different mitochondrial subtypes). The cloning efficiency and the development potential of these embryos was documented and differentially expressed candidate genes were screened among these groups, resulting in differentially expressed candidate gene library. .Based on our previous work, we will further explore the important functions of candidate genes and verify their functions at the cloned bovine embryos level. At the same time, we will study the epigenetic factors, such as histone deacetylase inhibitors TSA, Scriptaid, H3K9me3 demethylase exemplified by Kdm4d on their effectiveness in improving the efficiency of cloned cattle, and their role in nucleus-cytoplasm interrelationship on the development of cloned embryos. In turn, we hope to reveal some insight on the molecular mechanism of reprogramming, in the process of constructing the cytoplasmic and epigenetic regulation of network affecting the development of cloned animals, and thus providing a theoretical basis for understanding the molecular mechanism of somatic cell cloning.

应用体细胞克隆技术制备克隆牛不仅具有重要的经济价值，而且对于阐明牛克隆胚胎的重编程机理也具有重要意义。我们先前系统地比较和研究了来自同一个体的卵母细胞和供体细胞（同体）、不同个体的卵母细胞和供体细胞（异体）及同样线粒体单倍体（同型）和不同线粒体单倍体（异型）克隆的效率、发育潜能和不同类型克隆胚胎的转录组，筛选出了差异表达候选基因库。本项目拟在原有工作基础上，进一步深入探索候选基因的重要功能，并在克隆牛胚胎水平验证候选差异基因的功能。同时将研究组蛋白去乙酰化酶抑制剂TSA、Scriptaid、H3K9me3去甲基化酶Kdm4d等表观修饰因子在提高克隆牛效率，尤其是线粒体异型克隆胚胎发育潜能中的功能作用，鉴定其发育潜能提高后的表达模式及组蛋白甲基化和乙酰化状态的改变，揭示重编程的分子机制，构建克隆动物着床前胚胎发育过程中核质互作和表观遗传调控网络图，从而为阐明体细胞克隆的分子机理提供理论基础。

体细胞克隆技术在基因工程制药、动物遗传疾病模型建立以及动物资源保护和分子育种等方面都有着极其重要的应用价值。阐明牛克隆胚胎的重编程机理对提高克隆技术的成功率具有重要的指导意义。本项目在系统地比较和研究了卵母细胞和体细胞分别来自相同线粒体单倍体（同型）和不同线粒体单倍体（异型）时的克隆效率的基础上，深入地对比了同型、异型克隆胚胎以及自然受精胚胎的转录组，筛选出了各组间的差异表达候选基因库，并构建了克隆动物着床前胚胎发育过程中与核质互作和表观遗传调控相关的基因相互作用网络图。在此基础上，通过基因敲除和使用小分子抑制剂等手段验证了两个候选差异表达基因在早期胚胎发育过程中的重要功能，并对一个与表观修饰相关的候选差异表达基因的下游靶标化合物的代谢进行干预，获得了组蛋白H3K9me3这一重编程主要障碍完全擦除的供核细胞。项目还把研究延伸到了着床后的克隆胚胎，发现克隆后代由中胚层相关发育分化形成的器官（心脏和肾脏）常出现发育异常，鉴定了一个与中胚层发育分化相关的重要候选分子，同时探索了在新生死亡克隆牛存在端粒异常缩短的现象。这些研究结果为我们深入阐明重编程的分子机制并以此提高体细胞克隆成功率提供了理论基础。