Abstract
The ability to express exogenous cDNAs while suppressing endogenous genes via RNAi represents an extremely powerful research tool with the most efficient non-transient approach being accomplished through stable viral vector integration. Unfortunately, since traditional restriction enzyme based methods for constructing such vectors are sequence dependent, their construction is often difficult and not amenable to mass production. Here we describe a non-sequence dependent Gateway recombination cloning system for the rapid production of novel lentiviral (pLEG) and retroviral (pREG) vectors. Using this system to recombine 3 or 4 modular plasmid components it is possible to generate viral vectors expressing cDNAs with or without inhibitory RNAs (shRNAmirs). In addition, we demonstrate a method to rapidly produce and triage novel shRNAmirs for use with this system. Once strong candidate shRNAmirs have been identified they may be linked together in tandem to knockdown expression of multiple targets simultaneously or to improve the knockdown of a single target. Here we demonstrate that these recombinant vectors are able to express cDNA and effectively knockdown protein expression using both cell culture and animal model systems.
Highlights
The past decade has seen unprecedented technological and informational advances giving today’s researcher nearly unfettered access to genome sequences and transcriptome expression analysis of both diseased and normal tissue
To facilitate the process of generating such viruses we sought to create lentiviral expression vectors capable of expressing a cDNA and marker from bicistronic mRNA by modifying an existing commercial lentiviral vector, pLEX (OpenBiosystems). This self-inactivating [24,25] lentiviral expression vector was altered to contain a ccdB cassette flanked by 59 attR1 and 39 attR3 sites placed downstream of CMV promoter/enhancer sequences creating a Gateway-compatible Destination vector called pLEG(R1–R3) (Figure 1Aiii)
This vector was designed for use in three-plasmid recombination reactions with Entry vectors containing a cDNA between attL1-attL2 sites (Figure 1Ai) and genetic markers between attR2-attL3 sites (Figure 1Aii)
Summary
The past decade has seen unprecedented technological and informational advances giving today’s researcher nearly unfettered access to genome sequences and transcriptome expression analysis of both diseased and normal tissue. Retroviral and lentiviral vectors offer the ability to efficiently transfer genetic material to multiple cell types for longterm expression both in vitro as well as in vivo and in the case of lentiviruses, allow for non-dividing cells to be transduced [1,2]. These vectors represent an important ‘‘middle ground’’ between transient plasmid based systems and in vivo gene manipulation. There exist a number of commercial retroviral and lentiviral systems that allow cDNA overexpression or the use of RNA interference to ablate or diminish gene expression (as reviewed in [3,4])
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