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

Nucleic Acids Research
Stefan BergerDusan Bartsch

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 c...Continue Reading

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Citations

Oct 30, 2012·Nature·Jayakrishnan NandakumarThomas R Cech
Jul 20, 2011·Proceedings of the National Academy of Sciences of the United States of America·Piyush KhandeliaEugene V Makeyev
Apr 12, 2013·Proceedings of the National Academy of Sciences of the United States of America·Björn TewsDusan Bartsch
Mar 31, 2011·Nucleic Acids Research·Michael P GantierBryan R G Williams
Nov 3, 2010·Addiction Biology·Rainer SpanagelAndreas Zimmer
Sep 5, 2012·BMC Biology·Kai SchönigDusan Bartsch
Feb 22, 2014·Cellular and Molecular Life Sciences : CMLS·Gernot NeumayerMinh Dang Nguyen
Sep 9, 2016·Nucleic Acids Research·Marta MauriMarina Chekulaeva
Feb 6, 2014·The Journal of Biological Chemistry·Gayle M GordilloChandan K Sen
Sep 26, 2020·Nature Communications·Sebastian PilslGünter Mayer

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Methods Mentioned

BETA
transfection
gene knockdown
PCR
FCS
Assay
transgenic
Protein assay
flow cytometry
RNA Assay
xenograft

Software Mentioned

Image J

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