New Type Of Sirna Expression Vectors, Random Sirna Library Construction And Screening Based On A Random Sirna Library Cell Proliferation Related Genes,

RNAi has been widely used as a tool to study gene function in mammals since the discovery that synthetic siRNA duplexes can induce gene-specific RNAi acivity in mammalian cells in 2001, several small-scale RNAi screening with smaller siRNA libraries have also been done successfully. Among several siRNA expression vectors, short hairpin siRNA vector which use a polymerase (Pol) III promoter to drive the expression of short hairpin RNA is the most appealing one because of its high siRNA expression efficiency and relatively simple procedure of vector cloning. However, hairpin vectors still suffer from some limitations such as low-cloning-efficiency and being difficult to sequence, moreover, hairpin siRNA encoding sequence is hard to obtain, so generation of libraries in hairpin vectors is technically difficult and time-consuming. So far, the lack of high complexity siRNA libraries has been the main obstacle for large-scale RNAi screening in mammals. Herein, we generated a new type dual Pol III siRNA expression vector, in which the siRNA encoding sequence is common DNA instead of hairpin DNA, and therefore it will ravel out all the limitations brought on by hairpin structure. We also constructed a random siRNA library in this new type vector and used this random siRNA library to perform a phenotype-driven genomewide screening in a cell proliferation model.In our newly designed siRNA expression cassette, a gene-specific siRNA sequence is inserted between two convergent RNA Pol III promoters, then the sense and antisense strands of the siRNA duplex can be transcribed by these two promoters from the same template placed in between. After H1 and U6 promoters were modified at the -1 — -11 position to accommodate transcriptional terminator sequences, We constructed siRNA transient expression plasmid pDH (with two H1 promoters) , pBUH (with H1/U6 promoters) and two eukaryotic siRNA stable expression vector pCUH, pEG-UH ( both all with H1/U6 promoters), RNAi-knockdown of luciferase reporter genes and studies using a siQuant siRNA evaluation system showed that siRNA expressed by these series of new vectors could achieve equivalent RNAi efficiency to those expressed by classic hairpin siRNA-encoding vectors or chemically synthesized siRNAs. RNase-protection assay also...