利用胚胎干细胞特异性高表达MicroRNAs调控猪克隆胚胎基因组重编程的实验研究

Pig is an important organism both for bio-medical research and agricultural production. Currently, the clone technology based on somatic cell nuclear transfer (SCNT) is the main method for pig genetic manipulation. However, the low efficiency of this technology limits the development of novel genetically modified pigs for bio-medical or agricultural purposes. It is well established that somatic genomic reprogramming is the limitting factor determining the developmental potential of cloned embryos and the resulted clone efficiency. Very recently, it was reported that the microRNAs preferrently expressed in embryonic stem cells (ESC-specific microRNAs) were capable of rebustly and efficiently inducing pluripotent cells (iPSC) without trasncriptional factors, and the efficiency of somatic genomic reprogramming mediated by ESC-specific miRNAs was up to 10%, which was almost two order magnititude of that of the conventional method using transcription factors. In this project, we are planning to systematically evaluate the impacts of ESC-specific miRNAs, which contain different seed sequences and target different genes, on genomic repragramming and developmental potential of pig cloned embryos and the resulted overall clone efficieny, and then thereby to select the microRNAs or microRNA combinations with the highest efficiency. The completion of the work described in this project would laid down a foundation to establish a highly effieicent technological system for pig SCNT.

猪是重要的医学模型与经济动物。体细胞克隆是研制人类重大疾病转基因猪模型、培育重要经济性状转基因猪新品种的主要途径。现有猪体细胞克隆技术的低效率（一般低于1%）是制约转基因猪疾病模型与新品种培育规模化开展的主要因素，而如何促进克隆胚胎基因组在短时间内完成重编程是提高猪克隆效率的关键。最近研究表明，在胚胎干细胞中特异性高表达的microRNAs具备很强的快速介导体细胞基因组重编程的能力，其单独诱导形成iPS细胞的效率比传统4个转录因子（OSKM）高100倍以上，且诱导时间缩短1倍。本项目拟将含有不同"种子序列"（seed sequence）、针对不同靶基因的胚胎干细胞特异性microRNAs导入猪克隆胚胎，系统评估不同microRNA对猪克隆胚胎基因组重编程、发育能力以及猪克隆效率的影响，筛选适宜于猪克隆胚胎胞质微环境、效应最优的microRNA或其组合，为建立更高效的猪克隆技术体系奠定基础。

本项目的关键科学问题是评估胚胎干细胞特异性的microRNAs（miRNAs）对猪克隆胚胎基因组重编程的影响、筛选可促进猪克隆胚胎基因组重编程的胚胎干细胞特异性miRNAs，进而促进猪克隆胚胎基因组重编程、提供克隆猪培育效率;本项目的关键技术问题是建立高效的猪基因敲入技术，制备干细胞特异性基因位点敲入标记基因的转基因猪及其衍生细胞系，为准确评估胚胎干细胞特异性miRNAs对猪克隆基因组重编程的影响提供高活性的工具细胞和正常胚胎对照。.依据本项目同行评议建议及项目的原定计划，我们首先建立了高效的猪基因敲入技术体系。首次发现猪胚胎细胞具有很强的通过HDR（homology-directed repair）机制修复DSB的活性、进而实现高效率基因敲入的能力；基于此发现，我们研制了首例含有人同源点突变的基因编辑猪疾病新模型和首例血清白蛋白人源化转基因猪，首次通过CRISPR/Cas9诱导的HDR实现了外源基因在首建猪中100%的定点敲入率，并首次发现作为HDR模板的ssODN（single strand oligo DNA）高浓度时可抑制猪胚胎细胞中的HDR活性；此外，还研制了在alpha S1-Casein（alpha S1酪蛋白）基因位点敲入乳铁转运蛋白（LTF）的转基因猪和乳铁转运蛋白-溶菌酶（LTF-LYZ）的复合转基因猪，为解决仔猪哺乳期抗病性这一养猪业重要问题奠定了基础。.依托建立的高效基因敲入技术体系，开展了在Oct4基因位点终止密码子位点敲入标记基因EGFP的（Oct4-EGFP）转基因猪制备实验，目前已有一个受体成功受孕，有望获得OCT4与EGFP可视化共表达的转基因猪，进而为更准确的评估胚胎干细胞特异性miRNAs对猪克隆胚胎基因组重编程的影响提供高活性工具细胞和正常胚胎对照；利用中国科学院广州生物医药与健康研究所赖良学课题组惠赠的在Oct4基因终止密码子位点敲入了RFP（Oct4-RFP）的细胞为工具细胞，初步评估了胚胎干细胞特异性miRNAs对克隆胚胎基因组重编程的影响，发现胚胎干细胞特异性miRNAs的导入对克隆胚胎早期发育无显著影响，但可促进RFP的表达，提示胚胎干细胞特异性miRNAs可促进猪克隆胚胎的基因组重编程。