Journal of RNAi and Gene Silencing (2007), 3(1), 248-253

New Methods and Technologies

A strategy for constructing and verifying short hairpin RNA expression vectors

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

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

†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

© Copyright Fang-Jun Jia et al

(Received 21 October 2006; Revised 01 December 2006; Accepted 15 December 2006)