利用孤雄单倍体胚胎干细胞建立Tex101基因敲除小鼠及Tex101基因功能研究

Efficient and quick production of genome modified animals are much valuable for both basic and applied research. Recently great achievements have made on mammalian haploid stem cell studies. One finding is that the mouse androgenetic haploid embryonic stem (ahES) cells can take the place of sperm to produce healthy offspring after intracytoplasmic injectoin into oocytes, which provides a new approach to generate genome modified animals. However, due to loss of sperm epigenetic modifications during the establishment and culture of the cell lines, different ahES cell lines are variable in producing mice, and their developmental potential is largely reduced after long-term in vitro culture, which hinders the further application. In present study, we plan to evaluate the developmental potential of ahES cell lines. Through analysis of the epigenetic modifications of ahES cell lines with different developmental potential, we will try to find out the core epigenetic modifications that affect the development of ahES cell produced mice. And through small molecule screening and culture system modifications, we will improve the culture system which can better stabilize the core modifications. Tex101 gene may play important roles in reproduction. Next we will use ahES cells that are better maintained to generate Tex101 gene knockout mouse models, and further study the in vivo physiological role and regulatory mechanisms of Tex101 with the knockout mice. Our research will establish a stable platform for gene knockout mediated by ahES cells, and promote the application of this new technique in studies of gene function, reproduction and development.

建立高效、快捷的基因修饰动物制备体系，在基础和应用研究中都具有重要价值。近来哺乳动物单倍体干细胞研究取得突破进展。其中，小鼠孤雄单倍体胚胎干细胞在注射进卵胞质后能够替代精子完成发育，产生健康后代，为制备基因修饰动物提供了一种新的途径。然而由于建系和培养过程中精子表观修饰特性的丢失，不同孤雄单倍体胚胎干细胞系的发育能力不稳定，且在长期培养后大大降低，阻碍了其进一步的应用。在本项研究中，我们拟对孤雄单倍体干细胞系的发育能力进行评估，分析比较它们之间的基因表达和表观修饰差异，找到决定发育能力的核心表观修饰元件；通过小分子筛选和培养体系优化，建立可以稳定核心表观修饰的培养体系；进而利用单倍体干细胞途径建立在生殖过程中有潜在重要调控功能的Tex101基因敲除小鼠，阐明该基因的体内生理功能和调控机制。我们的研究将建立基于孤雄单倍体干细胞的高效稳定基因敲除平台，推动其在基因功能和生殖发育研究中的应用。

建立高效、快捷的基因修饰动物制备体系，在基础和应用研究中都具有重要价值。过往研究发现，小鼠孤雄单倍体胚胎干细胞在注射进卵胞质后能够替代精子完成发育，产生健康后代，为制备基因修饰动物提供了一种新的途径。然而由于建系和培养过程中精子表观修饰特性的丢失，不同孤雄单倍体胚胎干细胞系的发育能力不稳定，且在长期培养后大大降低，阻碍了其进一步的应用。在本项研究中，我们取得了以下研究成果：1）发现Tex101基因敲除雄性小鼠完全不育，并进一步揭示了Tex101调控的分子机制。2）早期胚胎发育是终末分化的配子通过重编程开启发育程序的必由之路。3）建立单倍体上胚层干细胞。4）建立Dlk1-Dio3印记区域的报告基因体系并用于干细胞多能性的快速指示和筛选。5）诱导多能性干细胞（iPS细胞）的免疫原性研究。我们的研究建立了基于单倍体干细胞的高效稳定基因敲除平台，推动其在基因功能和生殖发育研究中的应用。