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An optogenetic gene expression system with rapid activation and deactivation kinetics

Abstract

Optogenetic gene expression systems can control transcription with spatial and temporal detail unequaled with traditional inducible promoter systems. However, current eukaryotic light-gated transcription systems are limited by toxicity, dynamic range or slow activation and deactivation. Here we present an optogenetic gene expression system that addresses these shortcomings and demonstrate its broad utility. Our approach uses an engineered version of EL222, a bacterial light-oxygen-voltage protein that binds DNA when illuminated with blue light. The system has a large (>100-fold) dynamic range of protein expression, rapid activation (<10 s) and deactivation kinetics (<50 s) and a highly linear response to light. With this system, we achieve light-gated transcription in several mammalian cell lines and intact zebrafish embryos with minimal basal gene activation and toxicity. Our approach provides a powerful new tool for optogenetic control of gene expression in space and time.

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Figure 1: Model for the EL222-based light-inducible gene expression system.
Figure 2: Dose-dependent activation and photoreversibility of gene expression by VP-EL222.
Figure 3: Kinetic modeling of VP-EL222 activation.
Figure 4: Light-regulated gene expression of the splicing factor CELF2 using VP-EL222 in the T cell–derived JSL1 cell line.
Figure 5: VP-EL222 robustly activates reporter gene expression in the developing zebrafish embryo in a light-dependent manner.

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Acknowledgements

This work was funded by grants from the US National Institutes of Health (R01 GM081875 and GM106239 to K.H.G.; R01 GM103383 to K.W.L.; R01 GM096164 to O.D.W.), Cancer Prevention and Research Institute of Texas (RP130312), Defense Advanced Research Projects Agency (Living Foundries HR0011-12-C-0068 to B. Chow (University of Pennsylvania), supporting S.G.) and the Robert A. Welch Foundation (I-1424 to K.H.G.). K.H.G. is the Virginia Lazenby O'Hara Chair in Biochemistry and W.W. Caruth Scholar in Biomedical Research. A.R. was supported by a National Science Foundation Graduate Research Fellowship. We thank S.L. McKnight and P.R. Potts (both at UT Southwestern Medical Center) for generously providing constructs.

Author information

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Authors

Contributions

L.B.M.-M., A.R., O.D.W., K.W.L. and K.H.G. conceived and designed the experiments. L.B.M.-M., A.R. and M.J.M. performed the experiments. L.B.M.-M., A.R., M.J.M., O.D.W., K.W.L. and K.H.G. analyzed the data, with S.G. and K.H.G. generating the kinetic model. L.B.M.-M. and K.H.G. wrote the paper.

Corresponding author

Correspondence to Kevin H Gardner.

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Competing interests

L.B.M.M. and K.H.G. have filed US Patent Application PCT/US2012/065493 covering the method described in this paper.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Figures 1–5, Supplementary Table 1 and Supplementary Notes 1 and 3. (PDF 2701 kb)

Supplementary Note 2

MATLAB code for kinetic model (DOC 28 kb)

Supplementary Video 1

Z-stack of 70% epiboly embryo showing mosaic expression of mCherry after illumination with blue light. (AVI 20581 kb)

Supplementary Video 2

Z-stack of 70% epiboly embryo showing no expression of mCherry under dark conditions. (AVI 24709 kb)

Supplementary Video 3

Localization of fluorescent mCherry in the heart of a developing zebrafish embryo at 24 h.p.f. (AVI 399 kb)

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Motta-Mena, L., Reade, A., Mallory, M. et al. An optogenetic gene expression system with rapid activation and deactivation kinetics. Nat Chem Biol 10, 196–202 (2014). https://doi.org/10.1038/nchembio.1430

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