Genome-scale Screening in a Rat Haploid System Identifies Thop1 as a Modulator of Pluripotency Exit

Rat embryonic stem cells (ESCs), which are widely studied, can self-renew and exhibit pluripotency in long-term culture, but the mechanism underlying how they exit pluripotency remains obscure. Rat haploid ESCs (haESCs) enable advances in the discovery of unknown functional genes owing to their homozygous and pluripotent characteristics. Herein, we performed genome-wide mutation using piggyBac transposons in rat haESCs and obtained differentiation-retarded mutants assisted by Rex1-GFP reporter selection. High-throughput sequencing analysis further revealed numerous insertions related to various pathways affecting random differentiation. Thereafter, deletion of Thop1 (one candidate gene in the screened list) arrested the differentiation of rat ESCs by inhibiting the phosphorylation of ERK1/2, whereas overexpression of Thop1 promoted rat ESCs exit from pluripotency. Our findings provide an ideal tool to study functional genomics in rats: a homozygous haploid system carrying a pluripotency reporter that facilitates robust discovery of the mechanisms involved in the self-renewal or pluripotency of rat ESCs.大鼠胚胎干细胞（embryonic stem cells, ESCs）被广泛研究，其可在长期培养中自我更新并维持多能性，但其退出多能性的潜在机制仍不明确。大鼠单倍体胚胎干细胞（haploid embryonic stem cells, haESCs）因其兼具纯合性与多能性的特征，可推动未知功能基因的相关研究进展。本研究中，我们通过piggyBac转座子在大鼠单倍体ESCs中开展全基因组诱变，并借助Rex1-GFP报告基因筛选获得了分化阻滞突变体。高通量测序分析进一步揭示了大量与影响随机分化的多条信号通路相关的插入位点。随后，对筛选列表中的候选基因Thop1进行敲除，可通过抑制ERK1/2（extracellular regulated protein kinases 1/2）的磷酸化阻滞大鼠ESCs的分化；而过表达Thop1则会促进大鼠ESCs退出多能性。本研究的发现为大鼠功能基因组学研究提供了理想工具：一种携带多能性报告基因的纯合单倍体系统，可助力高效解析大鼠ESCs自我更新或多能性维持相关的分子机制。