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Construction of a long hairpin RNA expression library using Cre recombinase

Paper ID Volume ID Publish Year Pages File Format Full-Text
23853 43478 2012 11 PDF Available
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.

► We developed a method to generate a lhRNA expression library using Cre recombinase. ► We established a method for construction of a cDNA plasmid library. ► The lhRNA expression plasmids induced strong RNAi in a silkworm cell line.

Keywords
RNA interference; Library; Long hairpin RNA; Silkworm cell line; Cre recombinase
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Construction of a long hairpin RNA expression library using Cre recombinase
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 160, Issues 3–4, 31 August 2012, Pages 129–139
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us