title: A single lentiviral vector platform for microRNA-based conditional RNA interference and coordinated transgene expression.
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 Galpha12 and Galpha13 both singly and in combination, demonstrating the Galpha13 dependence of serum response element-mediated transcription. In RAW264.7 macrophages, regulated knockdown of Gbeta2 correlated with a reduced Ca(2+) response to C5a. Insertion of a GFP transgene upstream of the Gbeta2 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.
authors.affiliation: Alliance for Cell Signaling Molecular Biology Laboratory, California Institute of Technology, 1200 East California Boulevard, Pasadena, CA 91125, USA.
authors.forename: Kum-Joo
authors.initials: KJ
authors.lastname: Shin
authors.forename: Estelle A
authors.initials: EA
authors.lastname: Wall
authors.forename: Joelle R
authors.initials: JR
authors.lastname: Zavzavadjian
authors.forename: Leah A
authors.initials: LA
authors.lastname: Santat
authors.forename: Jamie
authors.initials: J
authors.lastname: Liu
authors.forename: Jong-Ik
authors.initials: JI
authors.lastname: Hwang
authors.forename: Robert
authors.initials: R
authors.lastname: Rebres
authors.forename: Tamara
authors.initials: T
authors.lastname: Roach
authors.forename: William
authors.initials: W
authors.lastname: Seaman
authors.forename: Melvin I
authors.initials: MI
authors.lastname: Simon
authors.forename: Iain D C
authors.initials: ID
authors.lastname: Fraser
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D044365:GTP-Binding Protein alpha Subunits, G12-G13 / Q000235:genetics; D018390:Gene Targeting / Q000379:methods*; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D023281:Genomics / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D006412:Hematopoietic Stem Cells / Q000187:drug effects / Q000378:metabolism; D016086:Lentivirus / Q000235:genetics; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D035683:MicroRNAs / Q000235:genetics / Q000494:pharmacology; D034622:RNA Interference; D027602:Serum Response Element / Q000235:genetics; D013752:Tetracycline / Q000494:pharmacology; D019076:Transgenes
chemical_list: D035683:MicroRNAs; D049452:Green Fluorescent Proteins; D044365:GTP-Binding Protein alpha Subunits, G12-G13; D013752:Tetracycline