A single lentiviral vector platform for microRNA-based conditional RNA interference and coordinated transgene expression
| Autor: | Estelle A. Wall, William E. Seaman, Jong Ik Hwang, Leah A. Santat, Melvin I. Simon, Robert A. Rebres, Jamie Liu, Iain D. C. Fraser, Joelle R. Zavzavadjian, Tamara I. A. Roach, Kum Joo Shin |
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| Jazyk: | angličtina |
| Rok vydání: | 2006 |
| Předmět: |
Transgene
Genetic Vectors Green Fluorescent Proteins Biology GTP-Binding Protein alpha Subunits G12-G13 Viral vector Small hairpin RNA Mice Transcription (biology) RNA interference microRNA Animals Transgenes Cells Cultured Gene knockdown Multidisciplinary Macrophages Lentivirus Gene targeting Genetic Therapy Genomics Fibroblasts Tetracycline Biological Sciences Hematopoietic Stem Cells Molecular biology Cell biology MicroRNAs Serum Response Element Gene Targeting RNA Interference Caltech Library Services |
| Popis: | 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 Ca 2+ 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. |
| Databáze: | OpenAIRE |
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