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Open Access Highly Accessed Methodology article

Modified gateway system for double shRNA expression and Cre/lox based gene expression

Nikolina Radulovich12, Lisa Leung13 and Ming-Sound Tsao123*

Author Affiliations

1 University Health Network, Ontario Cancer Institute/Princess Margaret Hospital Site, 610 University Ave., Toronto, Ontario, M5G 2M9, Canada

2 Department of Laboratory Medicine and Pathobiology, Faculty of Medicine, University of Toronto, Medical Sciences Buildings, 1 King's College Circle, Toronto, Ontario, M5 S 1A8, Canada

3 Department of Medical Biophysics, University Health Network, Ontario Cancer Institute/Princess Margaret Hospital Site, 610 University Ave., Toronto, Ontario, M5G 2M9, Canada

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BMC Biotechnology 2011, 11:24  doi:10.1186/1472-6750-11-24

Published: 22 March 2011

Abstract

Background

The growing need for functional studies of genes has set the stage for the development of versatile tools for genetic manipulations.

Results

Aiming to provide tools for high throughput analysis of gene functions, we have developed a modified short hairpin RNA (shRNA) and gene expression system based on Gateway Technology. The system contains a series of entry and destination vectors that enables easy transfer of shRNA or cDNA into lentiviral expression systems with a variety of selection or marker genes (i.e. puromycin, hygromycin, green fluorescent protein-EGFP, yellow fluorescent protein-YFP and red fluorescent protein-dsRed2). Our shRNA entry vector pENTR.hU6.hH1 containing two tandem human shRNA expression promoters, H1 and U6, was capable of co-expressing two shRNA sequences simultaneously. The entry vector for gene overexpression, pENTR.CMV.ON was constructed to contain CMV promoter with a multiple cloning site flanked by loxP sites allowing for subsequent Cre/lox recombination. Both shRNA and cDNA expression vectors also contained attL sites necessary for recombination with attR sites in our destination expression vectors. As proof of principle we demonstrate the functionality and efficiency of this system by testing expression of several cDNA and shRNA sequences in a number of cell lines.

Conclusion

Our system is a valuable addition to already existing library of Gateway based vectors and can be an essential tool for many aspects of gene functional studies.