Using A Novel CDNA-based RNA Interference Library To Identify Modulators Involving In Self-renewal Of Embryonic Stem Cells

Embryonic stem cells are excellent model for studying molecular mechanisms of cellular differentiation and development, and they have great potential in regenerative medicine. Gene knockout is one of the most important techniques for the study of gene function. However, this technique is technical demanding, labor-intensive and time-consuming that limits its routine use in the laboratory and it can not be used for large scale loss-of-function screening in embryonic stem cells. Furthermore, due to low frequency of homologous recombination in mammalian cells, it is difficult to achieve gene knockout in both alleles in embryonic stem cells. Although techniques such as microarray and differential display can be used for large scale identification of genes that are modulated at certain condition, the function of the genes still has to be determined by additional experiments in most of the cases. Direct functional screening at a high-throughput scale represents a new direction in genetic screening.RNA interference (RNAi) libraries have been proved to be a powerful tool for large scale functional genetic screens in model organisms such as Caenorhabditis elegans. However, their use in mammalian cells was not reported until recently. Since 2003, several RNAi libraries that can be used for loss-of-function screening in mammalian cells have been established. The first reported RNAi library consisted of synthesized siRNA duplexes that targeted 510 individual genes, most of them encoding kinases. A systematic screening of modulators of TRAIL-induced apoptosis with this library identified both known and unknown genes, which proved the worthiness of the RNAi library in functional genetic screens in mammalian cells. Subsequently, screening with several different RNAi libraries have identified new molecules involving many different functions, including tumor suppressors and drug targets, modulators of tumor cell motility, molecules involving signal transduction pathways. Most of RNAi libraries reported so far consisted of synthesized siRNAs or siRNA vectors that targeted genes with known sequences or non-coding RNAs. Although synthesized siRNAs transfected into mammalian cell had excellent knockdown efficiency, they are more costly and can not be used for functional screens that need long term inhibition of gene expression. RNAi libraries constructed in plasmids or viral vectors can be expanded easily and may provide long lasting knockdown of gene expression. The disadvantage of this kind of RNAi libraries is that tens of thousands individual siRNAs or siRNA vectors have to be produced, stored and used, making it costly and labor-intensive. A different approach is the construction of random RNAi libraries, either by insertion of enzyme-digested cDNA into RNAi vectors or by cloning of randomly synthesized 21bp DNA fragments. In theory, these libraries could target any gene in the mammalian cell. Considering the fact that the targeting sequence is only about 21nt long, it is very difficult to establish a high quality library that could cover most of the genes within mammalian cells.In this study, a new and simple strategy to produce siRNA expression vectors with two opposing polymerase III promoters was first developed. The design involved a one-step PCR amplification and a single cloning procedure to construct a dual promoter siRNA expression vector. Sense and antisense strands of siRNA duplex were transcribed from the same template and this eliminated the need to synthesize long hairpin-forming oligonucleotides. Our study demonstrated that this vector design could mediate potent inhibition of expression of both exogenous and endogenous genes in mammalian cells. It provides a simple and cost-effective method for rapid and large scale generation of siRNA vectors. It could also be applied to establish randomized RNAi library through random synthesis of the 19 to 21 siRNA encoding sequence of the reverse primer.Next, to facilitate high throughput functional genetic screens in embryonic stem cells, a simple and efficient system to construct cDNA-based random RNAi library was developed. Previous studies have demonstrated that sequence-specific gene silencing could be induced by long dsRNA in mouse embryos, mouse oocytes, embryonic stem cells and other mammalian cells. Based on these findings, a long 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 cDNA interference (cDNAi) library was established by cloning of enzyme-digested cDNA of mouse ES cells into BamHI site of convergent dual promoter RNAi vector. Sequencing of 20 randomly selected clones from the library showed that 17 contained inserts and all of them were unique sequence. A functional genetic screen of genes involving in self-renewal with the cDNAi library identified ubiquitin. The ubiquitin knockdown ES cell line generated 20%-30% of undifferentiated colonies in the absence of LIF, whereas parental ES cells and control vector pDCont transfectants produced less than 5% of colonies of undifferentiated cells, suggesting that ubiquitin play a role in ES cell differentiation.In conclusion, a novel approach to construct a cDNA-based random RNAi library was developed in the study and it provides a useful tool for investigation of molecular mechanisms of cellular differentiation and development. Our study has also proved that ubiquitin is a factor contributing to pluripotent phenotype of murine ES cells.