家兔体细胞核移植重编程机制的研究

This project is to study nuclear reprogramming of a somatic cell transferred into both oocyte and zygote, as a result that the imperfect reprogramming imposed by oocyte can be corrected by zygotic cytoplasm. Compared with other mammalian cloning, rabbit somatic cell nuclear transfer (SCNT) is more difficult with much lower efficiency. Fertilized egg possesses effective and orderly gene expression and regulation patterns, in turn, promotes the progression of normal embryo development. This study will establish the “recloning” nuclear transfer by which a somatic nucleus is transferred into an enucleated oocyte and develop into a cloned morulae after “first reprogramming”; the embryonic nuclei from NT embryo are subsequently transferred into mitotic metaphase enucleated zygote to complete “second reprogramming” which results in an improved reprogramming. We have carried a long term of rabbit SCNT study with a solid research background and expertise. We will study (1) the pattern of global DNA methylation and histone modification; (2) the expression of candidate genes, such as Oct 4, Nanog, Sox2 and their regulatory regions with regards to relative methylation and histone modification; (3) in search for new important molecules related to reprogramming by ChIP-Seq and RNA-Seq; (4) improving rabbit cloning efficiency. This study will explore new mechanism of nuclear reprogramming; pave the road toward large animal cloning, breeding and reproduction.

体细胞核移植是将分化的体细胞核移植到卵母细胞质中构建克隆胚经重编程发育成克隆动物。但卵母细胞重编程效率低，对细胞可塑性调控的机理不明, 克隆效率仅1-3%。受精卵具备有序的基因表达与调控，可激发胚胎的正常发育。我们积累了家兔核移植的理论与技能，培育了克隆兔。我们建立“再克隆”核移植技术，将家兔体细胞先后移入卵母细胞（MII）和中期受精卵（M期）重建再克隆胚。我们将探索不同卵细胞质调控细胞重编程的分子机理，研究受精卵矫正卵母细胞重编程不足的特定模式：（1）分析经两次重编程后克隆胚整体性DNA甲基化与组蛋白修饰的差异，（2）研究Oct4，Nanog，Sox2基因特定调控与表达的动态模式；（3）结合ChIP-Seq和RNA-Seq技术发现与重编程相关联的新分子与机制；（4）极大提高动物克隆的效率。家兔是重要的畜牧业经济物种和实验动物，本研究将为大家畜克隆、良种保存和快速繁育提供重要的理论基础。

体细胞核移植(SCNT)将高度分化的细胞重编程为具有发育全能性的胚胎细胞，产生克隆动物。但哺乳动物的克隆动物出生率极低，普遍仅1-3%。主要原因是重编程过程表观遗传障碍的存在，使卵母细胞胞质对体细胞核低效地修饰，重编程不完全，导致供体发育潜能降低。因此找到更多限制SCNT胚胎发育的重编程障碍对提高克隆效率具有重要意义，同时将有助于我们全面了解重编程机制。.首先检测了小鼠ZGA时期胚胎H3K9me3、H3K27me3、H3K4me3表观修饰动态模式，发现小鼠SCNT胚胎存在异常的高H3K9me3、H3K4me3修饰模式，而H3K27me3修饰呈现异常低的修饰模式。然后，在家兔胚胎中验证这些异常的组蛋白修饰是否具有进化的高保守性，发现家兔SCNT胚胎也存在高H3K9me3、H3K27me3的修饰模式，而H3K4me3修饰整体低于受精卵。.成功构建了受精卵-再克隆（二次克隆）技术体系，同时比较了二次克隆胚胎和一次克隆胚胎ZGA时期H3K9me3、H3K27me3、H3K4me3的修饰模式差异。发现二次克隆胚胎高H3K9me3修饰的现象得到了明显改善，恢复到了受精卵的修饰水平，表明受精卵的细胞质对SCNT胚胎的细胞核具有一定的重编程增强或矫正的作用。.小鼠克隆胚胎呈现异于受精卵的高H4K20me3修饰现象，甲基转移酶Suv4-20h2过度表达，而去甲基化酶Rad23b表达不足，是导致SCNT胚胎出现该障碍的原因，敲降Suv4-20h2降低克隆胚胎的H4K20me3水平，使其恢复到受精卵的水平，克服该障碍可以显著提高NT胚胎的发育能力以及克隆动物的出生率。表明H4K20me3是小鼠体NT重编程过程新的表观遗传障碍。.本研究有助于了解SCNT重编程过程中的表观遗传机制，为大动物家畜克隆、良种保存、濒危物种克隆保护、以及基于人的治疗性克隆应用的发展提供理论支持。