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Nucleic Acids Research Advance Access published online on September 22, 2006
Nucleic Acids Research, doi:10.1093/nar/gkl624
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© 2006 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

Inducible microRNA expression by an all-in-one episomal vector system

Alexey Epanchintsev, Peter Jung, Antje Menssen and Heiko Hermeking*

Molecular Oncology, Independent Max-Planck-Research Group, Max-Planck-Institute of Biochemistry, Am Klopferspitz 18 D-82152 Martinsried/Munich, Germany

*To whom correspondence should be addressed. Tel: +49 89 8578 2875; Fax: +49 89 8578 2540; Email: herme{at}biochem.mpg.de

Received August 7, 2006. Revised August 7, 2006. Accepted August 8, 2006.

Here we describe an episomal, one-vector system which allows the generation of cell populations displaying homogenous, inducible gene inactivation by RNA interference in a one step procedure. A dual tet-repressor/activator system tightly controls a bi-directional promoter, which simultaneously drives expression of microRNAs and a fluorescent marker protein. We demonstrate the effectiveness of this vector by knockdown of p53 expression in a human cell line which resulted in the expected loss of G1-arrest after DNA damage. The generation of a cell pool homogenously expressing the ectopic microRNAs was achieved in 1 week without the need for viral infections. Induction of microRNA expression did not elicit an interferon response. Furthermore, the vector was adapted for convenient ligation-free transfer of microRNA cassettes from public libraries. This conditional knockdown-system should prove useful for many research and gene therapeutic applications.

The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors


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P. Jung, B. Verdoodt, A. Bailey, J. R. Yates III, A. Menssen, and H. Hermeking
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