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

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