雄原核关键基因在小鼠胚胎发育和胚胎干细胞中的功能及机制研究

Normal fertilized eggs form a female pronucleus and a male pronucleus. Zygote with only male pronuclei can support embryonic development of somatic cell nuclear transfer, but zygote with only the female pronuclear fertilized egg loses the ability of reprogram somatic cells, indicating that some key reprogramming factors are specifically enriched in the male pronucleus. Our mass spectrometry analysis of male and female pronuclei revealed a number of proteins that were specifically enriched in each. However, there has been no systematic analysis of key genes in the male and female pronuclei and studies on function of these key genes in embryonic development and embryonic stem cells. In this project, the results of the proteomics of the male and female were analyzed and verified detailedly. We use androgenetic haploid embryonic stem cells as a model to construct a knockout cell library by CRISPR/Cas9 technology to study function of these key genes in pluripotency, haploidy maintenance, and epigenetics of embryonic stem cells. Furthermore, uncover the effects on the developmental potential of semi-cloned embryos after knockouts of these genes. This project has important scientific significance for understanding the mechanisms of early embryonic development and somatic cell reprogramming.

正常受精卵形成一个雌原核和一个雄原核。只具有雄原核的受精卵能支持体细胞核移植胚胎发育，而只具有雌原核受精卵丧失了体细胞重编程的能力，说明有些关键的重编程因子富集到了雄原核中。我们对雌雄原核进行了质谱分析发现了一些特异富集在雌雄原核中的蛋白。但是，目前并没有关于雌雄原核中关键基因的系统性分析和关键基因在胚胎发育以及胚胎干细胞中功能的研究。本研究项目中，拟对雌雄原核蛋白质谱数据进行详细分析和验证，以孤雄单倍体胚胎干细胞为模型，利用CRISPR/Cas9技术构建敲除的细胞文库，研究这些基因对于孤雄单倍体胚胎干细胞的多能性、单倍性维持和表观遗传组学方面的影响。同时，研究这些基因敲除后对半克隆胚胎发育潜能的影响。本研究项目对理解小鼠早期胚胎发育和重编程的调控机制具有重要科学意义。

本研究通过对小鼠胚胎雄原核富集蛋白因子进行分析，筛选了100个差异显著的关键基因进行在单倍体细胞和小鼠制作方面进行试验验证。首先，小鼠孤雄单倍体胚胎干细胞（AG-haESCs）和Cas9-sgRNA library的结合得到了很多基因敲除的半克隆小鼠，发现Med6可能是敲除致死的基因。受精卵胞质注射Cas9 mRNA和sgRNA，确认了Med6基因纯合敲除的小鼠死在E9.5-E10.5，杂合型可以正常的存活和繁殖后代。Gdf9-Cre与Loxp结合制作的卵源条件性敲除小鼠卵巢8周时重量只有WT的一半，体积约为1/4，卵巢中只有原始卵泡，没有其他阶段的卵母细胞存在。卵巢中卵母细胞的死亡发生在出生后第4-8周，尤其出生后第7周。3周卵巢中的GV只有50%处于NSN阶段。GV体外成熟发现，CKO组能够发育到M II阶段，但是其染色体散乱的排布在卵母细胞中。4周CKO母鼠的卵母细胞ICSI试验中，在E4.5天WT组89.5%的囊胚率，CKO组的囊胚率仅为4%，其余83.3%胚胎停在了1-细胞和2-细胞期。Med6基因敲除致死和影响小鼠早期胚胎发育，可能通过影响卵母细胞和卵泡发育过程中卵源基因的转录实现。总之，小鼠孤雄单倍体胚胎干细胞是基因功能筛选和研发新基因功能的快速高效新工具。