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

Shin, Kum-Joo and Wall, Estelle A. and Zavzavadjian, Joelle R. and Santat, Leah A. and Liu, Jamie and Hwang, Jong-Ik and Rebres, Robert and Roach, Tamara and Seeman, William and Simon, Melvin I. and Fraser, Iain D. C. (2006) A single lentiviral vector platform for microRNA-based conditional RNA interference and coordinated transgene expression. Proceedings of the National Academy of Sciences of the United States of America, 103 (37). pp. 13759-13764. ISSN 0027-8424. https://resolver.caltech.edu/CaltechAUTHORS:SHIpnas06

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Abstract

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.


Item Type:Article
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https://doi.org/10.1073/pnas.0606179103DOIUNSPECIFIED
Additional Information:Copyright © 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.
Subject Keywords:G protein; tetracycline
Issue or Number:37
Record Number:CaltechAUTHORS:SHIpnas06
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:SHIpnas06
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:4662
Collection:CaltechAUTHORS
Deposited By: Archive Administrator
Deposited On:02 Sep 2006
Last Modified:02 Oct 2019 23:15

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