OxfordOxford Mon - Fri 09:00-17:00 +44(0)1865-600222
LibPubMedia1@gmail.com
OxfordOxford Mon - Fri 09:00-17:00 +44(0)1865-600222
LibPubMedia1@gmail.com
J Venom Res
Open-access
Aptamers
Open-access
Aptamers 2019
03-04 April 2019, Oxford
Submit a manuscript

A strategy for constructing and verifying short hairpin RNA expression vectors

New Methods and Technologies

J RNAi Gene Silenc (May 2007), 3(1), 248-253

doi: jrgsxx

Published online: 23 January 2007

Full Text: (html | pdf ~571kb | refs)

A strategy for constructing and verifying short hairpin RNA expression vectors

Fang-Jun Jia †, Mei Huang †, Yuan-Chang Yan ‡, Yi-Ping Li †*

† Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China

‡ Model Organism Division, E-Institutes of Shanghai Universities, Shanghai, China

*Correspondence to: Yi-Ping Li, Email: oocyte@sunm.shcnc.ac.cn, Tel: +21 54921415 , ext 5951, Fax: +21 54921011

Received: 21 October 2006, Revised: 01 December 2006, Accepted: 15 December 2006

© Copyright The Authors

—————————————————————————————————————–

ABSTRACT

The application of RNA interference (RNAi) to study gene function is now commonplace in a variety of biological systems. Producing short hairpin RNA (shRNA) by DNA vectors is one popular strategy for RNAi applications. Here, we describe a one-step PCR method, termed reverse PCR, for constructing shRNA expression vectors. Characteristically, the pair of primers binds to circular plasmid in a back-to-back manner. The anchored primers provide the templates of shRNA sense strand and antisense strand locating to the two separate ends of PCR segment, which will benefit the PCR amplification and subsequent cloning by avoiding premature formation of a hairpin configuration. Finally, the establishment of a circular vector is achieved by self-ligation of the single PCR product. In addition, our results indicated that the hairpin loop including a single restriction site is resistant to digestion, while inclusion of twin restriction sites in the loop leads to activity, creating an optimal strategy for verifying sequences of shRNA template.

KEYWORDS: RNAi, cloning, reverse PCR, loop, vector, siRNA, shRNA, sequencing

—————————————————————————————————————–

Leave a Reply