雄原核因子促进体细胞克隆胚胎发育潜能的机制探索

Although somatic cell nuclear transfer can support full-term development and the derivation of nuclear transfer embryonic stem cell (ntESC) lines, the efficiency of animal cloning remains very low and the efficiency of derivation of ntESC lines is also lower than normal fertilized embryos. It is widely accepted that DNA methylation is critical for regulating gene expression in cloned embryos. Our previous study indicated that early-stage cloned mouse embryos possess significantly higher DNA methylation levels than the normal fertilized embryos. The factors resided in male pronucleus can efficiently erase DNA methylation of somatic nuclei. We further demonstrated that fusion of an additional male pronucleus can significantly improve the ability of in vivo development of cloned embryos. In the present proposal, we first aim to analyze the whole-genome DNA methylation levels of cloned embryos which are reconstructed with an addition or with reduced male pronucleus. Furthermore, we will investigate the gene expression characteristics of these cloned embryos to uncover the molecular mechanism that the male pronucleus can improve cloning efficiency. Finally, we will inject the mRNAs of male pronucleus-specific DNA demethylation related factors identified above into oocytes to validate the effects on cloned embryos development and the efficiency of ntESC lines derivation. Overall, we anticipate that our study can provide some novel clues to understand the molecular mechanism of somatic cell reprogramming and ultimately can enhance the development of cloned embryos greatly.

体细胞核移植可以获得核移植胚胎干细胞（ntESC）和克隆动物。但是目前克隆效率还很低，ntESC的建系效率也远低于正常受精胚胎。DNA甲基化是影响克隆胚胎基因表达和发育的关键因素之一，我们前期研究发现小鼠克隆胚胎DNA甲基化水平明显高于正常胚胎。而合子雄原核能有效去除供体细胞的DNA甲基化修饰，且增加一个雄原核可以显著提高克隆胚胎的体内外发育能力(Liu W et al. Cell Reports, 2014)。在本申请中我们将首先检测增加或去除雄原核的克隆胚胎DNA甲基化水平，并进一步检测其对克隆胚胎基因表达的影响。通过对基因组甲基化和表达的相关性分析，揭示雄原核提高克隆胚胎发育的分子机制。通过向卵母细胞中注射雄原核特异性的DNA去甲基化相关因子的mRNA，在DNA甲基化、基因组表达模式、克隆胚胎体内外发育和建立ntESC方面验证其对克隆胚胎发育的影响，为提高核移植效率提供理论基础。

体细胞核移植（somatic cell nuclear transfer, SCNT），是指将供体细胞核经显微操作移入去核的卵母细胞中，组成重构胚胎并使之发育成新动物个体的技术，是一种高效、快速地使细胞获得多能性的方式。然而，这个过程是非常低效的，大多数克隆胚胎阻滞在特定的发育阶段。通过对SCNT胚胎进行胚胎活检并结合单细胞测序，我们得到了具有不同发育命运胚胎的的全基因组DNA甲基化和RNA转录图谱。通过系统分析具有不同发育潜能的SCNT胚胎中差异表达基因的DNA甲基化状态和表达水平，我们发现Kdm4b和Kdm5b这两种组蛋白去甲基化酶未被激活，分别导致克隆胚胎在2-细胞阶段和4-细胞阶段停滞。SCNT过程中Kdm4b和Kdm5b异位表达可清除H3K9me3屏障，恢复转录谱，使囊胚发育效率提高95%以上。此外，这些克隆的胚胎可以更高效地进行核移植胚胎干细胞的诱导及克隆动物的出生。我们的研究表明，组蛋白和DNA甲基化重编程对SCNT胚胎的发育至关重要，为进一步完善治疗性克隆提供了重要线索。