title: A novel method to convert a DNA fragment inserted into a plasmid to an inverted repeat structure.
abstract: Transfection of an expression plasmid possessing inverted repeat (IR) DNA into cultured cells leads to the overexpression of hairpin RNA and efficient suppression of target gene expression. Such DNA vector-based RNA interference (RNAi) is widely used for characterizing genes of interest in cultured cell lines. In this study, we developed a new method to convert an inserted DNA fragment (IDF) in specially designed plasmid vectors into an IR structure by using nicking endonucleases and BcaBEST DNA polymerase. This method consists of the following steps: (1) linearization of the plasmid with a nick by using a restriction enzyme and a nicking endonuclease, (2) formation of the hairpin-loop DNA at the end near the IDF of the linearized plasmid, (3) insertion of a nick at the other end of the IDF by a nicking endonuclease, (4) execution of the strand displacement reaction from the nick to synthesize IR DNA, and (5) self-ligation of the linear double-stranded DNA. The IR DNA containing expression plasmids constructed by this method effectively induced target-specific RNAi in a silkworm cell line. We further established a method to purify expression plasmids containing IR DNA. Our new methods provide techniques for the construction of long hairpin RNA (lhRNA) expression plasmids for silencing specific genes in silkworms and other organisms, and offer a fundamental methodology for constructing an lhRNA expression library from a cDNA plasmid library.
authors.affiliation: Innate Immunity Research Unit, National Institute of Agrobiological Sciences, Ibaraki 305-8634, Japan.
authors.forename: Kazuya
authors.initials: K
authors.lastname: Tomimoto
authors.forename: Kosuke
authors.initials: K
authors.lastname: Fujita
authors.forename: Jun
authors.initials: J
authors.lastname: Ishibashi
authors.forename: Shigeo
authors.initials: S
authors.lastname: Imanishi
authors.forename: Minoru
authors.initials: M
authors.lastname: Yamakawa
authors.forename: Hiromitsu
authors.initials: H
authors.lastname: Tanaka
mesh_terms: D000818:Animals; D001483:Base Sequence; D001709:Biotechnology / Q000379:methods*; D012831:Bombyx / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D004247:DNA / Q000235:genetics*; D015723:Gene Library; D020868:Gene Silencing; D005822:Genetic Vectors / Q000235:genetics*; D055029:Inverted Repeat Sequences / Q000235:genetics*; D010957:Plasmids / Q000235:genetics*; D012313:RNA / Q000235:genetics; D034622:RNA Interference; D014162:Transfection
chemical_list: D012313:RNA; D004247:DNA