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Nucleic Acids Research Advance Access originally published online on July 16, 2008
Nucleic Acids Research 2008 36(16):e101; doi:10.1093/nar/gkn443
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Nucleic Acids Research, 2008, Vol. 36, No. 16 e101
© 2008 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

Intronically encoded siRNAs improve dynamic range of mammalian gene regulation systems and toggle switch

David Greber1, Marie Daoud El-Baba2 and Martin Fussenegger1,*

1Institute for Chemical and Bioengineering, ETH Zurich, HCI F115, Wolfgang-Pauli-Strasse 10, CH-8093 Zurich, Switzerland and 2Dèpartement Gènie Biologique, Institut Universitaire de Technologie, IUTA, 43 Boulevard du 11 Novembre 1918, F-69622 Villeurbanne Cedex, France

*To whom correspondence should be addressed. Tel: +41 44 633 3448; Fax: +41 44 633 1234; Email: fussenegger{at}chem.ethz.ch

Received March 27, 2008. Revised June 24, 2008. Accepted June 27, 2008.

Applications of conditional gene expression, whether for therapeutic or basic research purposes, are increasingly requiring mammalian gene control systems that exhibit far tighter control properties. While numerous approaches have been used to improve the widely used Tet-regulatory system, many applications, particularly with respect to the engineering of synthetic gene networks, will require a broader range of tightly performing gene control systems. Here, a generically applicable approach is described that utilizes intronically encoded siRNA on the relevant transregulator construct, and siRNA sequence-specific tags on the reporter construct, to minimize basal gene activity in the off-state of a range of common gene control systems. To demonstrate tight control of residual expression the approach was successfully used to conditionally express the toxic proteins RipDD and Linamarase. The intronic siRNA concept was also extended to create a new generation of compact, single-vector, autoinducible siRNA vectors. Finally, using improved regulation systems a mammalian epigenetic toggle switch was engineered that exhibited superior in vitro and in vivo induction characteristics in mice compared to the equivalent non-intronic system.


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