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

Kum Joo Shin, Estelle A. Wall, Joelle R. Zavzavadjian, Leah A. Santat, Jamie Liu, Jong Ik Hwang, Robert Rebres, Tamara Roach, William Seaman, Melvin I. Simon, Iain D.C. Fraser

Research output: Contribution to journalArticlepeer-review

239 Citations (Scopus)

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 Gα12 and Gα13 both singly and in combination, demonstrating the Gα13 dependence of serum response element-mediated transcription. In RAW264.7 macrophages, regulated knockdown of Gβ2 correlated with a reduced Ca2+ response to C5a. Insertion of a GFP transgene upstream of the Gβ2 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.

Original languageEnglish
Pages (from-to)13759-13764
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume103
Issue number37
DOIs
Publication statusPublished - 2006 Sep 12

Keywords

  • G protein
  • Tetracycline

ASJC Scopus subject areas

  • General

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