Abstract
BackgroundThe growing need for functional studies of genes has set the stage for the development of versatile tools for genetic manipulations.ResultsAiming 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.ConclusionOur 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.
Highlights
The growing need for functional studies of genes has set the stage for the development of versatile tools for genetic manipulations
Design of modified Gateway short hairpin RNA (shRNA)/gene expression system We utilized a combination of restriction-based cloning and highly versatile Gateway site-directed recombination technology for the construction of modified gene/shRNA expression system
The construction of pENTR. hU6.hH1 would allow for easy transfer of subcloned shRNA cassettes to different gateway based expression vectors, shRNA targeting of multiple genes, and the increased knockdown of a single target using multiple shRNA sequences
Summary
The growing need for functional studies of genes has set the stage for the development of versatile tools for genetic manipulations. In the era of whole genome sequencing and proteomics, there has never been a greater need to develop versatile tools for gene functional studies. Such studies necessitate a series of genetic manipulations including overexpression and/or downregulation of genes of interest either in in vivo and/or in vitro settings. The most successful RNAi libraries based on retroviral [4] and lentiviral expression [5,6] of shRNAs have been utilized for large-scale functional genomic screens [6].
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