[Genome-wide lentivector-based pooled shRNA library optimization]

We have optimized lentiviral vector constructs and cassettes for expression of short hairpin RNAs (shRNAs) in order to create genome-wide library capable of inhibition of full variety of human mRNAs. The vector optimization has resulted in 15-20-fold improvement in virus stock titers. We found that in the context of lentiviral vector the most effective structure for the shRNA is simple hairpin with 21 nucleotide stem. The shRNA-expressing lentiviral constructs contain choice of puro(R), copGFP or H-2K(k) selective markers. The efficiency of the optimized library was evaluated in experiments on screening of shRNAs that reactivate oncosuppressor p53 in HeLa cells. The cells contained reporter construct with p53-dependent expression of a fluorescent protein, which allows cytofluorimetric isolation of cell population with reactivated p53.