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J Venom Res
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Aptamers
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Aptamers 2019
03-04 April 2019, Oxford
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A streamlined sub-cloning procedure to transfer shRNA from a pSM2 vector to a pGIPZ lentiviral vector


Technology Report

J RNAi Gene Silencing (2010), 6(2), 411-415

doi: jrgsxx

Published online: 27 October 2010

Full Text: (html | pdf ~230kb | refs)

A streamlined sub-cloning procedure to transfer shRNA from a pSM2 vector to a pGIPZ lentiviral vector

Sara Ansaloni, Nadav Lelkes, Jonathan Snyder, Charles Epstein, Aditi Dubey and Aleister J Saunders*

Department of Biology, Drexel University, Philadelphia, PA, USA

*Correspondence toAleister Saunders, Email: Aleister.Saunders@Drexel.edu, Tel: +1 215 895 6772, Fax: +1 215 895 1273

Received: 26 August 2010, Revised: 12 October 2010, Accepted: 13 October 2010

© Copyright The Authors

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ABSTRACT

RNA interference (RNAi) is a widely used molecular biology technique to investigate the importance of specific genes in molecular pathways. Since mammalian cells are equipped with endogenous RNAi processing machinery, it has become common practice to transfect constructs that encode for short hairpin RNAs that are then cleaved to form the active RNAi sequences that bind to target mRNAs. Given the profit potential of this research approach, companies have developed retroviral libraries of shRNA constructs targeting the majority of the human genes. Recent technologic advances have allowed the rapid improvement of the vectors carrying the shRNA constructs while the silencing sequences remain the same. Therefore, sub-cloning of shRNA sequences from more obsolete vectors to newer vectors is a straightforward way to take advantage of newer delivery technologies. We describe here a streamlined procedure to transfer shRNA sequences from the pSM2 retroviral vector to a newer pGIPZ vector that is more stable, contains a GFP cassette and allows the preparation of high titer viral particles for transduction of cells and in vivo use. We demonstrate that our protocol provides a cost-effective and fast method to successfully sub-clone shRNA from a pSM2 retroviral vector to a pGIPZ lentiviral vector making it a useful tool for the investigators that have purchased pSM2 vectors in the past and wish now to upgrade their constructs by inserting them in more versatile vectors.

KEYWORDS: Sub-cloning, shRNA, RNA interference, lentivirus, retrovirus, pSM2, pGIPZ, pTRIPZ

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