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Published September 12, 2006 | Published + Supplemental Material
Journal Article Open

A single lentiviral vector platform for microRNA-based conditional RNA interference and coordinated transgene expression


RNAi is proving to be a powerful experimental tool for the functional annotation of mammalian genomes. The full potential of this technology will be realized through development of approaches permitting regulated manipulation of endogenous gene expression with coordinated reexpression of exogenous transgenes. We describe the development of a lentiviral vector platform, pSLIK (single lentivector for inducible knockdown), which permits tetracycline-regulated expression of microRNA-like short hairpin RNAs from a single viral infection of any naïve cell system. In mouse embryonic fibroblasts, the pSLIK platform was used to conditionally deplete the expression of the heterotrimeric G proteins G{alpha}12 and G{alpha}13 both singly and in combination, demonstrating the G{alpha}13 dependence of serum response element-mediated transcription. In RAW264.7 macrophages, regulated knockdown of G{beta}2 correlated with a reduced Ca2+ response to C5a. Insertion of a GFP transgene upstream of the G{beta}2 microRNA-like short hairpin RNA allowed concomitant reexpression of a heterologous mRNA during tetracycline-dependent target gene knockdown, significantly enhancing the experimental applicability of the pSLIK system.

Additional Information

© 2006 by the National Academy of Sciences. Freely available online through the PNAS open access option. Contributed by Melvin I. Simon, July 21, 2006 We thank colleagues in the AfCS for criticism and insight during the course of this study. We thank Greg Hannon for the pSM2 plasmid, provision of manuscripts before publication, and helpful discussions. This work was supported by contributions from public and private sources, including the National Institute of General Medical Sciences Glue Grant Initiative (U54 GM062114). K.-J.S. and J.-I.H. were supported by National Institutes of Health Grant R37 GM034236 (to M.I.S.). Author contributions: K.-J.S., M.I.S., and I.D.C.F. designed research; K.-J.S., E.A.W., J.R.Z., L.A.S., and J.L. performed research; K.-J.S., E.A.W., J.R.Z., L.A.S., J.L., J.-I.H., R.R., T.R., and W.S. contributed new reagents/analytic tools; K.-J.S., E.A.W., J.R.Z., and I.D.C.F. analyzed data; and I.D.C.F. wrote the paper. Conflict of interest statement: No conflicts declared. Supplementary figures 5 and 6 are included as separate files.

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Published - SHIpnas06.pdf

Supplemental Material - SHIpnas06fig5.pdf

Supplemental Material - SHIpnas06fig6.pdf


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September 13, 2023
October 23, 2023