Quantitative analysis of conditional gene inactivation using rationally designed, tetracycline-controlled miRNAs

Stefan M. Berger, Brigitte Pesold, Simone Reber, Kai Schönig, Annette J. Berger, Ina Weidenfeld, Jun Miao, Martin R. Berger, Oliver J. Gruss, Dusan Bartsch

Research output: Contribution to journalArticlepeer-review

22 Scopus citations

Abstract

The combination of RNA interference (RNAi) with the tetracycline-controlled transcription activation (tet) system promises to become a powerful method for conditional gene inactivation in cultured cells and in whole organisms. Here, we tested critical sequence elements that originated from miRNA mR-30 for optimal efficiency of RNAi-based gene knockdown in mammalian cells. Rationally designed miRNAs, expressed conditionally via the tet system, led to an efficient knockdown of the expression of both reporter genes and the endogenous mitotic spindle protein TPX2 in HeLa cells. Quantitative studies of the tet-controlled gene inactivation revealed that the residual expression of the target gene is an intrinsic attribute of all cells that cannot be eliminated either by increasing the miRNA to target mRNA ratio or by simultaneous expression of miRNAs targeting different sequences within the transcript. The kinetic analysis of the reversibility of the miRNA mediated knockdown suggests that the recovery of target gene expression is primarily driven by cell division. Our miRNA design provides a useful tool for conditional gene inactivation in combination with the RNA-polymerase II based tet system. The identified characteristics of the conditional RNAi-mediated knockdown need to be considered for its application in cell culture or in vivo.

Original languageEnglish (US)
Article numbergkq616
Pages (from-to)e168-e168
JournalNucleic acids research
Volume38
Issue number17
DOIs
StatePublished - Jul 17 2010

All Science Journal Classification (ASJC) codes

  • Genetics

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