Construction of a long hairpin RNA expression library using Cre recombinase

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• 作者：Kazuya Tomimoto ; Minoru Yamakawa ; Hiromitsu Tanaka
• 关键词：RNA interference ; Library ; Long hairpin RNA ; Silkworm cell line ; Cre recombinase
• 刊名：Journal of Biotechnology
• 出版年：2012
• 出版时间：31 August, 2012
• 年：2012
• 卷：160
• 期：3-4
• 页码：129-139
• 全文大小：1395 K

文摘

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.