父母源表观遗传差异对核移植重编程影响机制探究

Somatic cell nuclear transfer has great advantages in totipotency research, large animal breeding, animal disease model and therapeutic cloning. However, low developmental efficiency is the main problem in nuclear transfer reprogramming. At present, the development of single cell sequencing technology has provided a reliable way to improve the efficiency of nuclear transfer. Therefore, the application of new sequencing technology in the development of nuclear transfer is likely to provide new clues and evidence for improving the efficiency of nuclear transfer.It is well known that the epigenetic modification barrier of nuclear transfer include H3K9me3, H3K4me3 and X chromosome inactivation. These epigenetic modifications show parental modification differences in normal fertilized eggs. Combined with our related work, abnormal epigenetic modification of parental differentiation is likely to be an important reason for the impediment of nuclear transfer. In this study, Multi-temporal and multi-dimensional sequencing of mouse nuclear transfer embryos was performed by novel low-input sequencing. Emphasis was placed on abnormalities of differential methylation region represented by imprinting genes. The new epigenetic disorders were found by modifying the abnormal areas in nuclear transfer reprogramming. And then we could find a new epigenetic modification disorder with parental differentiation and enrich the theory that epigenetic modification of parental differentiation is a nuclear transfer reprogramming barrier.. Finally, using the experience of mouse research, a preliminary attempt was made in primates to provide new directions and evidence for improving the efficiency of nuclear transfer in large animals.

体细胞核移植在全能性塑造、动物模型制备及治疗性克隆等方面具有巨大优势。然而发育效率低下限制其发展。目前，单细胞测序技术已为提高核移植效率提供可靠途径。因此，新型测序技术在核移植中的应用极可能为其效率提高提供新的途径。目前已知核移植表观修饰障碍包括H3K9me3、H3K4me3及X染色体失活等。这些表观修饰在正常受精卵里都具有父母源修饰差异。结合我们的相关工作，父母源差异化的表观修饰异常很有可能是核移植阻碍的重要原因。本研究中，利用新型低样本量测序对小鼠核移植胚胎进行多时程、多维度的测序。重点关注以印记基因为代表的差异甲基化区异常。并通过对异常区域修饰找到核移植重编程过程中新的表观遗传障碍。进而找到新的父母源差异化的表观遗传修饰障碍，丰富父母源差异化表观修饰是核移植重编程障碍的理论。最后，利用小鼠研究中的经验，在灵长类动物中进行初步测序，为大动物核移植效率提高提供新的方向和证据。

本研究主要研究核移植重编程过程中，父母源表观遗传修饰差异对核移植发育效率的影响。本研究内容主要通过利用测序技术揭示小鼠胚胎发育过程中的多项异常。主要包括：①建立小鼠父母源差异化表观遗传及染色体构象全景描绘。②发现小鼠差异基因或基因区域，并实现区域内的干预验证其对核移植重编程的发育影响。.③依据小鼠经验实现灵长类的表观遗传及染色体构象全景描绘。在项目执行过程中，该项目利用RNA-seq技术以及新型的三代测序技术建立起了部分差异表观遗传全景描绘并分析出了依赖于H3K27me3的非经典印记是核移植重编程障碍的重要影响因素。随后对该种表观遗传修饰影响的基因或染色体进行了基因改造从而实现了大幅度提升核移植重编程效率的新方法。验证了这种异常是核移植重编程的重要障碍。对于灵长类的表观遗传及染色体构象由于疫情影响，灵长类动物价格在过往三年大幅上涨，随后通过对猪及大鼠的物种描绘进行替代，亦描绘出在胚胎发育过程中大鼠及猪两个物种的染色体或表观遗传构象，实现了对灵长类表观遗传构象的替代。该计划执行期中，该研究出色了完成项目在预设过程中的计划。.