Optimization of a genome-wide disordered lentivector-based short hairpin RNA library |
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| Authors: | O A Guryanova M Makhanov A A Chenchik P M Chumakov E I Frolova |
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| Institution: | (1) Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, 117997, Russia;(2) Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA;(3) System Biosciences (SBI), Mountain View, California, 94041, USA;(4) Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 119991, Russia |
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| Abstract: | 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. |
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| Keywords: | RNA interference functional genomics short hairpin RNA copGFP H-2Kk flow cytometry pseudovirus particle packing p53 genome-wide library efficient screening |
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