Abstract
Objectives:
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.
Materials and methods:
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.
Results:
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.
Conclusions:
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.