title: An inducible T7 RNA polymerase-dependent plasmid system.
abstract: RNA interference (RNAi) has become a powerful tool for the specific silencing of gene transcription. Especially the targeting of genes in mammalian cells has been greatly improved by generating plasmid based and viral vector-based systems. This permits expression of short hairpin RNA (shRNA) on a longterm basis. However, an inducible expression of shRNA is required, if the target is essential for cell survival. We developed a doxycycline-inducible two-plasmid system for the expression of a ribozyme-processed shRNA. In contrast to other existing systems, we use the highly specific T7 phage RNA polymerase, which does not interact with cellular factors; therefore, interference with cellular functions is limited. One plasmid is responsible for doxycycline-dependent expression of T7 RNA polymerase and a second plasmid expresses a ribozyme-processed shRNA under the control of a T7 promoter. Our results showed that doxycycline- dependent expression of T7 RNA polymerase was tightly controlled and expression of an shRNA against firefly luciferase inhibited 86% of luciferase activity. In conclusion, our plasmid system provides a very useful tool for analyzing essential gene functions in vitro.
authors.affiliation: Department of Medical Biotechnology, Paul-Ehrlich-Institute, Paul-Ehrlich-Str. 51-59, D-63225 Langen, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Hamdorf
authors.forename: Heide
authors.initials: H
authors.lastname: Muckenfuss
authors.forename: Ulrich
authors.initials: U
authors.lastname: Tschulena
authors.forename: Stephan
authors.initials: S
authors.lastname: Pleschka
authors.forename: Ralf
authors.initials: R
authors.lastname: Sanzenbacher
authors.forename: Klaus
authors.initials: K
authors.lastname: Cichutek
authors.forename: Egbert
authors.initials: E
authors.lastname: Flory
mesh_terms: D001483:Base Sequence; D012321:DNA-Directed RNA Polymerases / Q000235:genetics* / Q000378:metabolism*; D004318:Doxycycline / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D017930:Genes, Reporter / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics*; D012313:RNA / Q000096:biosynthesis / Q000235:genetics; D016337:RNA, Catalytic / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism*
chemical_list: D016337:RNA, Catalytic; D014764:Viral Proteins; D012313:RNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D004318:Doxycycline