A cDNA-Based Random RNA Interference Library for Functional Genetic Screens in Embryonic Stem Cells

Abstract

Abstract To facilitate high-throughput functional genetic screens in embryonic stem cells, a simple and efficient system to construct cDNA-based random RNA interference (RNAi) library was developed in the study. Previous studies have demonstrated that sequence-specific gene silencing could be induced by long double-stranded RNA (dsRNA) in mouse embryos, mouse oocytes, embryonic stem cells, and other mammalian cells. Based on these findings, a dsRNA-expressing RNAi vector system was designed. This study provided evidence that the vector design could induce efficient knockdown of expression of both exogenous egfp gene and endogenous MTM1 gene in mouse embryonic stem cells. A random RNAi library was established by cloning enzyme-digested cDNA of mouse embryonic stem (ES) cells into the BamHI site of the convergent dual promoter RNAi vector. Sequencing of 20 randomly selected clones from the library showed that 17 contained inserts and that all of them were unique sequences. A functional genetic screen of genes involving in self-renewal and differentiation with the random RNAi library identified ubiquitin. The ubiquitin knockdown ES cell line generated 20%–30% of undifferentiated colonies in the absence of leukemia inhibitor factor, whereas parental ES cells and control vector pDCont transfectants produced less than 5% of colonies of undifferentiated cells, suggesting that ubiquitin plays a role in ES cell differentiation. The random RNAi library provides a useful tool for investigation of molecular mechanisms of cellular development and differentiation.