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Biotechnol Bioeng. 2015 Jul;112(7):1483-7. doi: 10.1002/bit.25562. Epub 2015 Mar 13.

An optogenetic upgrade for the Tet-OFF system.

Author information

1
Faculty of Biology, University of Freiburg, Schänzlestrasse 1, 79104, Freiburg, Germany.
2
BIOSS Centre for Biological Signalling Studies, University of Freiburg, Schänzlestrasse 18, 79104, Freiburg, Germany.
3
Faculty of Biology, University of Freiburg, Schänzlestrasse 1, 79104, Freiburg, Germany. wilfried.weber@biologie.uni-freiburg.de.
4
BIOSS Centre for Biological Signalling Studies, University of Freiburg, Schänzlestrasse 18, 79104, Freiburg, Germany. wilfried.weber@biologie.uni-freiburg.de.

Abstract

The rapid development of mammalian optogenetics has produced an expanding number of gene switches that can be controlled with the unprecedented spatiotemporal resolution of light. However, in the "pre-optogenetic" era many networks, cell lines and transgenic organisms have been engineered that rely on chemically-inducible transgene expression systems but would benefit from the advantages of the traceless inducer light. To open the possibility for the effortless upgrade of such systems from chemical inducers to light, we capitalized on the specific Med25VBD inhibitor of the VP16/VP64 transactivation domain. In a first step, we demonstrated the efficiency and selectivity of Med25VBD in the inhibition of VP16/VP64-based transgene expression systems. Then, we fused the inhibitor to the blue light-responsive B-LID degron and optimized the performance of this construct with regard to the number of Med25VBD repeats. This approach resulted in an optogenetic upgrade of the popular Tet-OFF (TetR-VP64, tetO7 -PhCMVmin ) system that allows tunable, blue light-inducible transgene expression in HEK-293T cells.

KEYWORDS:

TET system; gene switch; inducible expression; optogenetics

PMID:
25683779
DOI:
10.1002/bit.25562
[Indexed for MEDLINE]

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