title: Construction of a long hairpin RNA expression library using Cre recombinase.
abstract: A large number of genome-wide screens using RNA interference (RNAi) libraries have been utilized to determine the function of individual gene products involved in a variety of biological processes. In this study, we describe a new method to enzymatically generate a long hairpin RNA (lhRNA) expression library from a cDNA plasmid library using a nicking endonuclease, BcaBEST DNA polymerase, and Cre recombinase without excising the inserted DNA fragment from the plasmid vector. This method involves 5 steps: (1) conversion of an inserted DNA fragment in a plasmid into a direct repeat (DR); (2) purification of the plasmid containing the DR; (3) subcloning a lox71 cassette into the plasmid; (4) conversion of the DR in the plasmid into an inverted repeat (IR) using Cre recombinase; and (5) purification of the plasmid containing the IR. We also established an efficient method for inserting DNase I-digested DNA fragments into expression plasmids to enable construction of a cDNA plasmid library suitable as source materials to construct the lhRNA expression library. We confirmed that each of the lhRNA expression plasmids constructed using this method induced strong RNAi in a silkworm cell line, NIAS-Bm-oyanagi2.
authors.affiliation: Insect Mimetics Research Unit, National Institute of Agrobiological Sciences, 1-2 Owashi, Tsukuba, Ibaraki 305-8634, Japan.
authors.forename: Kazuya
authors.initials: K
authors.lastname: Tomimoto
authors.forename: Minoru
authors.initials: M
authors.lastname: Yamakawa
authors.forename: Hiromitsu
authors.initials: H
authors.lastname: Tanaka
mesh_terms: D003001:Cloning, Molecular / Q000379:methods*; D015723:Gene Library; D019426:Integrases / Q000235:genetics*; D012313:RNA / Q000096:biosynthesis* / Q000235:genetics*
chemical_list: D012313:RNA; C045073:Cre recombinase; D019426:Integrases