Enzymatic production of RNAi libraries from cDNAs.
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.
Shirane
Daisuke
D
Department of Pharmacology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Sugao
Kohtaroh
K
Namiki
Shigeyuki
S
Tanabe
Mao
M
Iino
Masamitsu
M
Hirose
Kenzo
K
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*
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