Abstract
RNA interference (RNAi) is a conserved posttranscriptional gene silencing mechanism that has recently emerged as a breakthrough genetic tool in functional genomics and drug target discovery. An increasing number of studies applying RNAi in high-throughput screens have begun to unravel complex signaling networks underlying diverse cellular processes. This chapter describes an approach to construct a conditional small-hairpin (sh)RNA library and its application in human lymphoma cell lines. A library cloning procedure outlines the incorporation of shRNA sequences and random 60-mer “bar code” oligonucleotides, enabling rapid identification of the hairpin by microarrays. Lymphoma cell lines are optimized for efficient retroviral transduction and tetracycline inducibility. The shRNA library is suitable for identifying molecular targets in cancer, but also versatile for various screening strategies.
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Acknowledgments
I would like to thank Xin Yu, Hong Zhao, Laurence Lamy, Haihua Chu, Jenny Zhang, Cailin Collins, Weihong Xu, and Yandan Yang for the construction of the library. I thank Liming Yang, Wenming Xiao, John Powell, and George Wright for bioinformatics and statistical supports and R. Eric Davis for the FLYRD18/mCAT-IRES-Bleo cell line and packaging vectors. I am grateful to the generous support and helpful advice from Louis M. Staudt. This work was supported in part by the Damon Runyon Cancer Research Foundation to V.N.N. and the Intramural Research Program of the National Institute of Health, National Cancer Institute, to L.M.S.
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Ngo, V.N. (2013). Identification of Pathogenetically Relevant Genes in Lymphomagenesis by shRNA Library Screens. In: Küppers, R. (eds) Lymphoma. Methods in Molecular Biology, vol 971. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-269-8_14
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DOI: https://doi.org/10.1007/978-1-62703-269-8_14
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Publisher Name: Humana Press, Totowa, NJ
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