title: Enzymatic production of RNAi libraries from cDNAs.
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.
authors.affiliation: Department of Pharmacology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Shirane
authors.forename: Kohtaroh
authors.initials: K
authors.lastname: Sugao
authors.forename: Shigeyuki
authors.initials: S
authors.lastname: Namiki
authors.forename: Mao
authors.initials: M
authors.lastname: Tanabe
authors.forename: Masamitsu
authors.initials: M
authors.lastname: Iino
authors.forename: Kenzo
authors.initials: K
authors.lastname: Hirose
mesh_terms: D002135:Calcium-Binding Proteins; D018076:DNA, Complementary / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000235:genetics; D003850:Deoxyribonuclease I / Q000378:metabolism; D015723:Gene Library; D017930:Genes, Reporter; D005822:Genetic Vectors; D006801:Humans; D019169:Jurkat Cells; D008840:Microfilament Proteins; D010957:Plasmids; D034622:RNA Interference / Q000502:physiology*; D034741:RNA, Small Interfering / Q000502:physiology*
chemical_list: C112986:AIF1 protein, human; D002135:Calcium-Binding Proteins; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D008840:Microfilament Proteins; D034741:RNA, Small Interfering; C085693:DNASE1 protein, human; D003850:Deoxyribonuclease I