State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai University, Tianjin, China.
Chongqing Key Laboratory of Human Embryo Engineering, Chongqing Health Center for Women and Children, Chongqing, China.
Cell Prolif. 2022 Apr;55(4):e13209. doi: 10.1111/cpr.13209. Epub 2022 Mar 11.
The rats are crucial animal models for the basic medical researches. 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. To investigate the key modulators on pluripotency exiting in rat ESCs, we perform genome-wide screening using a unique rat haploid system.
Rat haploid ESCs (haESCs) enable advances in the discovery of unknown functional genes owing to their homozygous and pluripotent characteristics. REX1 is a sensitive marker for the naïve pluripotency that is often utilized to monitor pluripotency exit, thus rat haESCs carrying a Rex1-GFP reporter are used for genetic screening. Genome-wide mutations are introduced into the genomes of rat Rex1-GFP haESCs via piggyBac transposon, and differentiation-retarded mutants are obtained after random differentiation selection. The exact mutations are elucidated by high-throughput sequencing and bioinformatic analysis. The role of candidate mutation is validated in rat ESCs by knockout and overexpression experiments, and the phosphorylation of ERK1/2 (p-ERK1/2) is determined by western blotting.
High-throughput sequencing analysis reveals numerous insertions related to various pathways affecting random differentiation. Thereafter, deletion of Thop1 (one candidate gene in the screened list) arrests the differentiation of rat ESCs by inhibiting the p-ERK1/2, whereas overexpression of Thop1 promotes rat ESCs to 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.
大鼠是基础医学研究中至关重要的动物模型。大鼠胚胎干细胞（ESCs）是广泛研究的对象，它们可以在长期培养中自我更新并表现出多能性，但它们退出多能性的机制仍不清楚。为了研究大鼠 ESCs 中多能性退出的关键调节因子，我们使用独特的大鼠单倍体系统进行全基因组筛选。
大鼠单倍体 ESCs（haESCs）由于其纯合和多能性特征，能够在发现未知功能基因方面取得进展。REX1 是一种用于监测多能性退出的原始多能性的敏感标记物，因此使用携带 Rex1-GFP 报告基因的大鼠 haESCs 进行遗传筛选。通过 piggyBac 转座子将全基因组突变引入大鼠 Rex1-GFP haESCs 的基因组中，并在随机分化选择后获得分化延迟突变体。通过高通量测序和生物信息学分析阐明确切的突变。通过 knockout 和过表达实验在大鼠 ESCs 中验证候选突变的作用，并通过 Western blot 测定 ERK1/2 磷酸化（p-ERK1/2）。
高通量测序分析揭示了许多与影响随机分化的各种途径相关的插入。此后，Thop1（筛选列表中的一个候选基因）的缺失通过抑制 p-ERK1/2 阻止大鼠 ESCs 的分化，而过表达 Thop1 则促进大鼠 ESCs 退出多能性。
我们的研究结果为研究大鼠功能基因组学提供了理想的工具：一种携带多能性报告基因的纯合单倍体系统，该系统有助于发现大鼠 ESCs 自我更新或多能性所涉及的机制。
