Modulation of gene expression by lentiviral-mediated delivery of small interfering RNA.
RNA interference (RNAi) mediates sequence-specific posttranscriptional gene silencing triggered by double-stranded RNA. RNAi is an effective tool for functional genomics in C. elegans, whereas biochemical characteristics of RNAi hamper its use in mammalian cells. We here analyze kinetic and quantitative aspects of mammalian RNAi in different cell lines using novel lentiviral constructs with double H1-shRNA expression cassettes located in the U3 region of the LTR. Using enhanced green fluorescence protein (EGFP) as a target gene for RNAi and red fluorescence protein (RFP) as surrogate marker for intracellular siRNA expression, we show that long-term siRNA expression mediates stable RNAi. Furthermore, RNAi-induced gene silencing varies from minimal to complete loss-of-function phenotypes within homogeneous and between different cell populations. Interestingly, the extent of gene silencing correlates to lentiviral integrations as well as siRNA expression levels in target cells. Finally, we demonstrate functional gene silencing of a cytokine receptor gene in normal CD34+ hematopoietic progenitor cells. These data suggest that the use of suitable lentiviral constructs to quantify siRNA expression in living cells may allow analysis of loss-of-function phenotypes in a dose-dependent manner in a wide variety of target cells.
Scherr
Michaela
M
Zentrum Innere Medizin, Abteilung Hämatologie und Onkologie, Medizinische Hochschule Hannover, Carl-Neuberg Strasse 1, D-30623 Hannover, Germany. M.Scherr@t-online.de
Battmer
Karin
K
Ganser
Arnold
A
Eder
Matthias
M
D000818:Animals; D018952:Antigens, CD34 / Q000235:genetics; D002460:Cell Line; D006224:Cricetinae; D015967:Gene Expression Regulation, Viral / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000378:metabolism; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D008164:Luminescent Proteins; D051379:Mice; D010641:Phenotype; D034622:RNA Interference / Q000502:physiology*; D034741:RNA, Small Interfering / Q000235:genetics*; D018121:Receptors, Cytokine / Q000235:genetics; D000097573:Red Fluorescent Protein
D018952:Antigens, CD34; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D034741:RNA, Small Interfering; D018121:Receptors, Cytokine; D000097573:Red Fluorescent Protein