单倍体维持因子的筛选和鉴定

For most metazoan cells, their genomes are diploid and they produce daughter cells via mitosis. But diploid primordial germ cells can generate haploid gametes through meiosis. Genomic imprinting and dosage compensation restrict haploid mammalian development to very early embryos. Recently, the haploid embryonic stem cells(haESC) were established successfully from mouse embryos, which encourage us to reinvestigate the haploid development and cell differentiation. Moreover, since haploid cells possess one copy of each gene, it is quite easy for us to generate the loss-of-function mutations in haESCs. Recessive mutations can also be assessed in forward genetic screens based on haESC. But until now, due to rapid self-diploidization in culture, the haESCs cannot maintain the haploid state for a long time without cell sorting. So, we aim to identify and characterize the “haploidy maintaining factors”. . In this study, we will create a genome-wide mutant library by transfecting the mouse haESCs with piggyBac transposon-based gene trapping vector. After high-throughput genetic screens on the library, the candidate genes involved in haploidy maintenance can be identified through Splinkerette-PCR combined with massively parallel sequencing. Furthermore, a small mutant library will be generated by using CRISPR/Cas9 genome editing system to validate the candidate genes. The function and underlying mechanism of these genes in haploidy maintaining will be investigated. Then, we will explore the possibility of establishing a stable haploid cell line and optimize the culture conditions . . Many reports have observed that the haploid state progressively lost in very early stage of embryonic development. Also, haESCs show strong preference for diploidization during lineage specification. Hence, the ploidy changes of haESC are closely related with some intrinsic mechanisms of development process. Therefore, the study of the mechanism of ploidy maintenance will enable us to deeply understand the development process and a variety of biological events of the organism. Due to the evolutionary conservation, we also expect that our study can provide guidance for the successful establishment of human haESC, which should be an invaluable tool in biomedical research in the future.

后生动物的绝大多数体细胞基因组是二倍体并以有丝分裂的方式繁殖，而原始生殖细胞经过减数分裂生成单倍体的配子细胞。基因印记和剂量补偿效应使哺乳动物单倍体的发育停滞在胚胎早期。近年来，单倍体胚胎干细胞（haploid ESC, haESC）的成功分离和建系引起了广泛关注，让我们重新思考胚胎发育和细胞分化的基本问题。但haESC在培养过程中会发生自发的二倍体化，不能长期维持单倍体状态。在本研究中，我们将寻找并鉴定“单倍体维持因子”。首先利用小鼠haESC和转座子基因捕获载体构建全基因组范围的突变体文库，通过高通量遗传筛选和二代测序技术获知与单倍体维持相关的候选基因；其次用CRISPR/Cas9基因编辑技术构建小型突变体文库来确认候选基因；并深入研究它们在倍性维持和变化过程中的作用机制；最终建立长期稳定维持单倍体状态的细胞系，并为人haESC的成功建立提供指导。

哺乳动物单倍体细胞只拥有一套染色体组，在遗传筛选和转基因动物培育中具有重要的价值。但是在haESC培养传代过程中，有一定比例的单倍体细胞会自发转变为二倍体细胞，限制了其应用。为了解决这个问题，我们将基于piggyBac（PB）转座子的插入基因突变载体导入到小鼠单倍体胚胎干细胞（haESC）中，构建了一个全基因组范围的纯合突变体文库。进而对文库进行传代培养并通过多轮流式分选富集单倍体细胞后，提取基因组并进行PB转座子插入位点的检测，发现Etl4基因突变体在细胞群体中明显富集。随后，在另外两株haESCs系中，用CRISPR/Cas9技术敲除Etl4基因后，发现它们的自发二倍化速率均明显下降，其多能性和自我更新能力则不受影响，表明Etl4的缺失可以帮助维持haESCs的单倍体状态，并且没有细胞系特异性。为探究Etl4缺失促进haESCs维持单倍体状态的机制，我们通过转录组RNA-Seq分析发现Etl4缺失使得细胞能量代谢相关基因表达水平发生显著变化，进而通过Seahorse代谢实验也验证了其OXPHOS水平上升、糖酵解水平下降的特殊能量代谢表型。随后，在培养过程中通过改变细胞能量代谢状态来维持haESCs的单倍体状态，发现添加促进OXPHOS的药物1,25-羟基维生素D3（1,25(OH)2D3），或者抑制糖酵解的药物三溴丙酮酸（3-BrPA），均可以让多株haESCs的单倍体细胞比例在较长时间内维持较高水平。该研究揭示了haESCs的自发二倍化与细胞能量代谢存在关联，并建立了一种通过调节培养基成分维持单倍体的方法，也为其它物种haESCs培养提供了新的思路。