biostudies-literatureGuryanova OANIA NIH HHSNCI NIH HHS396-405https://www.ebi.ac.uk/biostudies/studies/S-EPMC2748426biostudies-literatureUnknown40(3)To obtain a whole genome library that suppresses the total diversity of human mRNAs, lentiviral vector constructs and a short hairpin RNA (shRNA) expression cassette were optimized. The optimization of the vector increased the virus titer in preparations by 15-20 times. A simple shRNA structure with a 21-bp stem proved to be the most effective. Lentivector-based shRNA expression constructs were obtained by using puro(R), copGFP, or H-2K(k) as a selectable marker. The efficiency of the optimized library was demonstrated when screening for shRNAs reactivating the tumor suppressor p53 in HeLa cells. Cells carried a reporter construct ensuring p53-responsive synthesis of a fluorescent protein, which allowed selection of cells with reactivated p53 by flow cytometry.Molecular biologyOptimization of a Genome-Wide Disordered Lentivector-Based Short Hairpin RNA Library.PMC2748426R01 CA104903-02R01 AG025278-01A1R01 CA104903R01 AG025278Chenchik AAMakhanov MChumakov PMFrolova EIGuryanova OAfalseOptimization of a Genome-Wide Disordered Lentivector-Based Short Hairpin RNA Library.To obtain a whole genome library that suppresses the total diversity of human mRNAs, lentiviral vector constructs and a short hairpin RNA (shRNA) expression cassette were optimized. The optimization of the vector increased the virus titer in preparations by 15-20 times. A simple shRNA structure with a 21-bp stem proved to be the most effective. Lentivector-based shRNA expression constructs were obtained by using puro(R), copGFP, or H-2K(k) as a selectable marker. The efficiency of the optimized library was demonstrated when screening for shRNAs reactivating the tumor suppressor p53 in HeLa cells. Cells carried a reporter construct ensuring p53-responsive synthesis of a fluorescent protein, which allowed selection of cells with reactivated p53 by flow cytometry.2006-01-01T00:00:00Z2006 May2020-11-19T08:16:41Z2020-10-29T10:50:30ZS-EPMC27484261977712910.1134/S002689330603006X10.1134/s002689330603006x