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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Original Russian Text © O.A. Guryanova, M. Makhanov, A.A. Chenchik, P.M. Chumakov, E.I. Frolova, 2006, published in Molekulyarnaya Biologiya, 2006, Vol. 40, No. 3, pp. 448–459.
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Guryanova, O.A., Makhanov, M., Chenchik, A.A. et al. Optimization of a genome-wide disordered lentivector-based short hairpin RNA library. Mol Biol 40, 396–405 (2006). https://doi.org/10.1134/S002689330603006X
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DOI: https://doi.org/10.1134/S002689330603006X