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

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 appl...

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Bibliographic Details
Published in:Nucleic acids research 2008-09, Vol.36 (16), p.e101-e101
Main Authors: Greber, David, El-Baba, Marie Daoud, Fussenegger, Martin
Format: Article
Language:English
Subjects:
Animals
beta-Glucosidase - biosynthesis
beta-Glucosidase - genetics
Cell Line
CHO Cells
CRADD Signaling Adaptor Protein - biosynthesis
CRADD Signaling Adaptor Protein - genetics
Cricetinae
Cricetulus
Epigenesis, Genetic
Gene Expression Regulation
Genes, Reporter
Genetic Engineering - methods
Genetic Vectors
HeLa Cells
Humans
Introns
Methods Online
Mice
RNA Interference
RNA, Small Interfering - biosynthesis
RNA, Small Interfering - genetics
Trans-Activators - biosynthesis
Trans-Activators - genetics
Transgenes
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Summary: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.
ISSN:0305-1048
1362-4962
DOI:10.1093/nar/gkn443