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 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.
Greber
David
D
Institute for Chemical and Bioengineering, ETH Zurich, HCI F115, Wolfgang-Pauli-Strasse 10, CH-8093 Zurich, Switzerland.
El-Baba
Marie Daoud
MD
Fussenegger
Martin
M
D000818:Animals; D016466:CHO Cells; D053420:CRADD Signaling Adaptor Protein / Q000096:biosynthesis / Q000235:genetics; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D044127:Epigenesis, Genetic; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D007438:Introns; D051379:Mice; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000096:biosynthesis* / Q000235:genetics; D015534:Trans-Activators / Q000096:biosynthesis / Q000235:genetics; D019076:Transgenes; D001617:beta-Glucosidase / Q000096:biosynthesis / Q000235:genetics
D053420:CRADD Signaling Adaptor Protein; D034741:RNA, Small Interfering; D015534:Trans-Activators; C021514:cyanogenic beta-glucosidase; D001617:beta-Glucosidase