单细胞转录组测序的猪克隆胚胎发育相关母源重编程因子的鉴定及其功能研究

The premise of normal development of cloned embryos is the successful transformation of epigenetic modification patterns from differentiated donor cell to pluripotent embryo driven by the enucleated oocyte. Studies have suggested that maternal reprogramming factors can regulate the reconstruction of epigenetic modification pattern in cloned embryos, determining the developmental competence of cloned embryos. However, the role of maternal reprogramming factors in the development of cloned embryos is still poorly studied, and no study has been carried out to explore maternal reprogramming factors according to the developmental competence of cloned embryos derived from these oocytes and elucidate their mechanism. Accordingly, on the basis of preliminary research, this study will apply single cell transcriptome sequencing to explore and screen maternal reprogramming factors really related to the development of porcine cloned embryos, further investigate the effects of overexpression or knockdown of newly discovered candidate reprogramming factors on the development of cloned embryos, epigenetic modification reconstruction, the regulation of key gene expression pattern and DNA damage response in cloned embryos, and finally uncover the molecular mechanism underlying the developmental competence of cloned embryos regulated by newly discovered reprogramming factors. The discussion and clarification of this topic will discover new reprogramming factors determining the developmental competence of cloned embryos and promote the development of the research in nuclear reprogramming induced by somatic cell nuclear transfer and the application of somatic cell nuclear transfer in livestock and regenerative medicine.

克隆胚胎顺利发育的前提是去核的卵母细胞能够使供体细胞核的表观遗传修饰模式由分化状态转变为胚胎多能性状态。研究表明，母源重编程因子能够调控克隆胚胎表观遗传修饰模式重建，决定了克隆胚胎发育潜能。但目前对母源重编程因子调控克隆胚胎发育机制的认识还远远不够，而且也没有研究针对具有不同克隆胚胎发育潜能的卵母细胞进行重编程因子挖掘及阐明其作用机制。据此，本研究拟在前期工作基础上采用单细胞转录组测序来挖掘并筛选真正与猪克隆胚胎发育相关的母源重编程因子，继而利用过表达和干扰技术来阐明新发现的候选重编程因子在克隆胚胎发育、表观遗传修饰模式重建、关键基因表达模式调控和DNA损伤反应过程中的作用，最终揭示新发现的重编程因子调控克隆胚胎发育潜能的分子机制。相信该课题的探讨和阐明不仅能够挖掘到新的决定克隆胚胎发育潜能的重编程因子，而且将会对体细胞核移植重编程的机理研究及其在畜牧业和再生医学中的应用起到重要推动作用。

体细胞核移植是一种将供体动物细胞的细胞核移植到去核卵母细胞中，产生与供体细胞动物遗传物质一样的克隆动物的繁殖技术，在基础研究、畜牧产业及生物医学等领域具有重要价值，但目前低的克隆效率限制了其应用。本项目通过对不同发育能力的卵母细胞进行单细胞转录组测序，明确了30个差异表达基因；建立了胚胎小孔培养体系，明确了表观遗传修饰基因、印记基因和X染色体失活基因在不同发育潜能克隆胚胎及卵裂球中的表达模式；着重探讨了Dnmt1对克隆胚胎发育的影响，研究发现，克隆胚胎中扰低Dnmt1能够改善DNA甲基化重编程和基因表达来提高胚胎发育，进一步研究显示，Dnmt1o在卵母细胞成熟过程中稳定表达，扰低后，卵母细胞胞质成熟受到破坏，而Dnmt1s在克隆胚胎中DNA甲基化和表达紊乱，扰低后，关键基因DNA甲基化重编程、表达和克隆胚胎得到改善，明确了供体细胞中Dnmt1s是体细胞核移植DNA甲基化重编程的阻碍因子；分析了印记基因对克隆胚胎发育的影响，研究发现，克隆胚胎中基因组印记不能有效维持，供体细胞中印记紊乱会造成克隆胚胎发育能力降低，同时，克隆胚胎中调控印记基因能够改善基因组印记、胚胎凋亡和基因表达，提高了克隆胚胎发育；进一步探讨了5-aza-dC对克隆胚胎发育的影响，研究发现，供体细胞和克隆胚胎双重处理为最佳改善胚胎发育处理方式，处理后，核重塑增强，胚胎凋亡降低，DNA甲基化重编程和基因表达得到改善；还研究了褪黑激素对克隆胚胎发育的影响，发现，褪黑激素改善了胚胎发育进程，抑制了胚胎发育阻滞，促进了核重塑，抑制了胚胎凋亡，改善了克隆胚胎DNA甲基化重编程和基因表达，提高了克隆胚胎质量和发育。本项目阐明了调控克隆胚胎发育的关键因素，通过表观遗传修饰提高了体细胞核移植效率，对体细胞核移植技术在基础研究、畜牧业及再生医学等领域中的应用起到了重要推动作用。