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Enzymatic production of RNAi libraries from cDNAs

Nature Genetics volume 36, pages 190–196 (2004)Cite this article

Abstract

RNA interference (RNAi) induced by small interfering (siRNA) or short hairpin RNA (shRNA) is an important research approach in mammalian genetics. Here we describe a technology called enzymatic production of RNAi library (EPRIL) by which cDNAs are converted by a sequence of enzymatic treatments into an RNAi library consisting of a vast array of different shRNA expression constructs. We applied EPRIL to a single cDNA source and prepared an RNAi library consisting of shRNA constructs with various RNAi efficiencies. High-throughput screening allowed us to rapidly identify the best shRNA constructs from the library. We also describe a new selection scheme using the thymidine kinase gene for obtaining efficient shRNA constructs. Furthermore, we show that EPRIL can be applied to constructing an RNAi library from a cDNA library, providing a basis for future whole-genome phenotypic screening of genes.

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Figure 1: Outline of EPRIL.
Figure 2: Silencing of GFP expression by RNAi library prepared from cDNA encoding GFP.
Figure 3: RNAi efficiency profiles with various transduction protocols.
Figure 4: Silencing of type 1 IP3R expression by RNAi library derived from IP3R cDNA.
Figure 5: Thymidine kinase–based system for selection of efficient RNAi constructs.

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Acknowledgements

We thank A. Hashimoto for technical advice regarding FL5.12 cell culture and K. Serizawa for help in data analysis. This work was supported in part by Grants-in-Aid for Scientific Research and the Advanced and Innovational Research program in Life Sciences from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

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Author notes

  1. Daisuke Shirane and Kohtaroh Sugao: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Pharmacology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan

    Daisuke Shirane, Kohtaroh Sugao, Shigeyuki Namiki, Mao Tanabe, Masamitsu Iino & Kenzo Hirose

Authors
  1. Daisuke Shirane
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  2. Kohtaroh Sugao
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  3. Shigeyuki Namiki
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  4. Mao Tanabe
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Correspondence to Kenzo Hirose.

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Shirane, D., Sugao, K., Namiki, S. et al. Enzymatic production of RNAi libraries from cDNAs. Nat Genet 36, 190–196 (2004). https://doi.org/10.1038/ng1290

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