Conditional RNAi Using the Lentiviral GLTR System.
RNA interference (RNAi) has become an essential technology for functional gene analysis. Its success depends on the effective expression of target gene-specific RNAi-inducing small double-stranded interfering RNA molecules (siRNAs). Here, were describe the use of a recently developed lentiviral RNAi system that allows the rapid generation of stable cell lines with inducible RNAi based on conditional expression of double-stranded short hairpin RNA (shRNA). These lentiviral vectors can be generated rapidly using the GATEWAY recombination cloning technology. Conditional cell lines can be established by using either a two-vector system in which the regulator is encoded by a separate vector or by a one-vector system. The available different lentiviral vectors for conditional shRNA expression cassette delivery co-express additional genes that allow (1) the use of fluorescent proteins for color-coded combinatorial RNAi or monitoring RNAi induction (pGLTR-FP), (2) selection of transduced cells (pGLTR-S), and (3) the generation of conditional cell lines using a one-vector system (pGLTR-X).
Division of Molecular Pathophysiology, Biocenter, Innsbruck Medical University, Innrain 80/82, Innsbruck, Austria.
Elisabeth
E
Pfeiffenberger
Division of Molecular Pathophysiology, Biocenter, Innsbruck Medical University, Innrain 80/82, Innsbruck, Austria.
Reinhard
R
Sigl
Division of Molecular Pathophysiology, Biocenter, Innsbruck Medical University, Innrain 80/82, Innsbruck, Austria. stephan.geley@i-med.ac.at.
Stephan
S
Geley
D005786:Gene Expression Regulation / Q000235:genetics; D020868:Gene Silencing; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics
D034741:RNA, Small Interfering
GATEWAY; Human cell line; Inducible; Knockdown; Lentivirus; RNAi; Tetracycline; shRNA