Multipurpose modular lentiviral vectors for RNA interference and transgene expression.
We have created a multipurpose modular lentiviral vector system for expressing both transgenes and miRNA 30-based short hairpins (shRNAmirs) for RNAi. The core of the resulting vector system, pLVmir, allows a simple two step cloning procedure for expressing shRNAmirs under the control of a Pol II promoter in both a constitutive and conditional manner. The adapted cloning method includes a PCR-free method for transferring shRNAmir based RNAi clones from a publicly available library (Open Biosystems). The addition of a Pol II promoter-driven shRNAmir cassette and broadening the choice of Pol III promoters and silencing triggers offers great flexibility to this system. The combination of several preexisting and additional modules created here caters to common needs of researchers. Our modular vector system was validated regarding functionality of promoters, inducibility and reversibility. We successfully applied the system to knockdown Xirp2 mRNA expression in H2kb-tsA58 muscle cells and determined that this had no spurious effect on the expression of a closely related protein. Finally, our set of lentiviral vectors may be used to achieve synergistic effects, for simultaneous knockdown of two genes, as a rescue plasmid and for studying mutant proteins in a physiological context.
Department of Molecular Cell Biology, Institute for Cell Biology, University of Bonn, Ulrich-Haberland-Str. 61a, 53121 Bonn, Germany.
Venu
V
Kesireddy
Peter F M
PF
van der Ven
Dieter O
DO
Fürst
D002460:Cell Line; D005434:Flow Cytometry; D015870:Gene Expression; D055785:Gene Knockdown Techniques; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D019076:Transgenes / Q000235:genetics*
D034741:RNA, Small Interfering