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。

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.