An inducible T7 RNA polymerase-dependent plasmid system.
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.
Hamdorf
Matthias
M
Department of Medical Biotechnology, Paul-Ehrlich-Institute, Paul-Ehrlich-Str. 51-59, D-63225 Langen, Germany.
Muckenfuss
Heide
H
Tschulena
Ulrich
U
Pleschka
Stephan
S
Sanzenbacher
Ralf
R
Cichutek
Klaus
K
Flory
Egbert
E
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*
D016337:RNA, Catalytic; D014764:Viral Proteins; D012313:RNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D004318:Doxycycline