An inducible T7 RNA polymerase-dependent plasmid system.
Mol Biotechnol
; 33(1): 13-21, 2006 May.
Article
em En
| MEDLINE
| ID: mdl-16691002
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.
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Base de dados:
MEDLINE
Assunto principal:
Plasmídeos
/
Proteínas Virais
/
RNA Polimerases Dirigidas por DNA
/
Expressão Gênica
Idioma:
En
Ano de publicação:
2006
Tipo de documento:
Article