提高诱导绵羊iPSC效率的miRNAs筛选与评价

Abstract：The establishment of ovine embryonic stem cell（ESC） is very important for the generation of economically relevant gene-modified sheep. However, ovine ESCs are still not available, which limit the generation of precise gene-modified sheep. The establishment of induced pluripotent stem cell（iPSC） technology hold great promise for solving the problem and can use the iPSC instead of ESC to generate transgenic animals. Recently the ovine iPSC have been reported, but the reprogramming efficiency is still very low and lack of evidence for germ line contribution. miRNAs are posttranscriptional modulators of gene expression and play an important role in many developmental processes. Researches showed that embryonic stem cell specific miRNAs can promote induced pluripotency. On the basis of the ovine iPSC induction platform, we will study as below: 1. screening the superior candidate miRNA or miRNAs combination for improve the efficiency of reprogramming of sheep somatic cells; 2. Partially Study the function of these miRNAs in the reprogram procedure. This study will not only establish a rapid and highly efficient iPSC reprogramming system but also provide a platform to explore the culture conditions for ovine iPSC and ESC.

绵羊胚胎干细胞（ESC）系的建立对获得与经济性状相关的基因修饰绵羊至关重要。然而，迄今为止，真正的绵羊ESC系尚未获得。诱导多能性干细胞（iPSC）技术的建立，对解决这一问题提供了新的契机，能利用iPSC代替ESC来生产基因修饰绵羊。目前，国内外研究绵羊iPSC虽已有报道，但重编程效率很低，获得的iPSC未证明能进行生殖系传递获得可育后代。miRNA是基因表达的转录后调节器，研究证明胚胎干细胞特异性的miRNAs能促进体细胞重编程。本课题组前期初步建立了诱导绵羊iPSC的平台，课题组拟在前期工作的基础上：1、筛选出促进绵羊体细胞重编程的miRNA或miRNAs组合；2、初步探索这些miRNA在重编程过程中的作用，优化绵羊iPSC诱导方案。该课题的开展有望建立一种高效获得绵羊iPSC的体系，有助于提高克隆效率，为获得真正的绵羊iPSC和ESC奠定基础。

绵羊胚胎干细胞（ESC）系的建立对获得与经济性状相关的基因修饰绵羊至关重要。然而，迄今为止，真正的绵羊ESC系尚未获得。诱导多能性干细胞（iPSC）技术的建立，对解决这一问题提供了新的契机，能利用iPSC代替ESC来生产基因修饰绵羊。目前，国内外研究绵羊iPSC虽已有报道，但重编程效率很低，获得的iPSC未证明能进行生殖系传递获得可育后代。miRNA是基因表达的转录后调节器，研究证明胚胎干细胞特异性的miRNAs能促进体细胞重编程。本项目完成3种绵羊体细胞分离及诱导成siPSC等工作，建立了8种限定性因子+p53干扰+过表达ASF1A高效诱导绵羊体细胞为siPSCs的方法；使用Solexa高通量测序完成正常绵羊体细胞和siPSCs的miRNAs差异表达谱；筛选获得诱导过程中添加miR-302a/b/c/d-367、miR-429-200a-200b和miR-290等能显著性增加siPSC诱导效率；研究发现miR-302a/b/c/d-367能靶向于E2F家族转录因子，抑制其表达而激活内源Sox2、Oct4和Nanog等iPSC标记蛋白的表达；建立了dCas9-SAM系统上调内源的限定性因子表达，提高绵羊体细胞重编程的效率。本项目的研究将为iPSC诱导效率的提高提供理论依据，也为获得真正的绵羊iPSC和ESC奠定基础。