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The pluripotency of mouse embryonic stem (ES) cells is maintained by self‐renewal. To screen for genes essential for this process, we constructed an RNA interference (RNAi) library by inserting subtracted ES cell cDNA fragments into plasmid containing two opposing cytomegalovirus promoters. ES cells were transfected with individual RNAi plasmids and levels of the pluripotency marker  Oct‐4  were monitored 48 hours later by real time RT‐PCR. Of the first 89 RNAi plasmids characterized, 12 downregulated  Oct‐4  expression to less than 50% of the normal level and 7 of them upregulated  Oct‐4  expression to more than 150% of the normal level. To investigate their long‐term effect on self‐renewal, ES cells were transfected by these 19 RNAi plasmids individually and G418‐resistant colonies were subjected to alkaline phosphatase (AP) staining after 7 days selection. Except for 4 plasmids that caused cell death, the ratio of AP positive colonies was repressed to less than 60% of the control group by the other 15 plasmids and even below 20% by 10 plasmids. The cDNA fragments in these 10 plasmids correspond to eight genes, including  Zfp42/Rex‐1 , which was chosen for further functional analysis. RNAi knockdown of  Zfp42  induced ES cells differentiate to endoderm and mesoderm lineages, and overexpression of  Zfp42  also caused ES cells to lose the capacity of self‐renewal. Our results indicate that RNAi screen is a feasible and efficient approach to identify genes involved in ES cells self‐renewal. Further functional characterization of these genes will promote our understanding of the complex regulatory networks in ES cells.

Screening for Genes Essential for Mouse Embryonic Stem Cell Self‐Renewal Using a Subtractive RNA Interference Library