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Control of gene expression using a red- and far-red light–responsive bi-stable toggle switch

Nature Protocols volume 9, pages 622–632 (2014)Cite this article

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Abstract

Light-triggered gene expression systems offer an unprecedented spatiotemporal resolution that cannot be achieved with classical chemically inducible genetic tools. Here we describe a protocol for red light–responsive gene expression in mammalian cells. This system can be toggled between stable ON and OFF states by short pulses of red and far-red light, respectively. In the protocol, CHO-K1 cells are transfected to allow red light–inducible expression of the secreted alkaline phosphatase (SEAP) reporter, and gene expression is tuned by illumination with light of increasing wavelengths. As a starting point for elaborate red light–responsive gene expression, we outline the reversible activation of gene expression and describe how a spatial pattern can be 'printed' on a monolayer of cells by using a photomask. The core protocol requires only 4 d from seeding of the cells to reporter quantification, and other than light-emitting diode (LED) illumination boxes no elaborate equipment is required.

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Figure 1: Design of the red light–inducible transgene expression system.
Figure 2: Absorbance spectrum of PCB in methanol:HCl (95:5).
Figure 3: Setup of LED illumination boxes.
Figure 4: Adjustable gene expression by illumination with various wavelengths.
Figure 5: Switch-off kinetics.
Figure 6: Space-resolved gene expression.

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Acknowledgements

This work was supported by contract research 'Internationale Spitzenforschung II' of the Baden-Württemberg Stiftung (P-LS-SPII/2), by the European Research Council (ERC) under the European Community′s Seventh Framework Programme (FP7/2007-2013)/ERC grant agreement no. 259043-CompBioMat, and by the Excellence Initiative of the German Federal and State Governments (EXC 294). We thank J. Schmidt, D. Schächtele and J. Meßmer (University of Freiburg) for designing and constructing the illumination boxes.

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Authors and Affiliations

  1. Faculty of Biology, University of Freiburg, Freiburg, Germany

    Konrad Müller, Matias D Zurbriggen & Wilfried Weber

  2. Centre for Biological Signalling Studies (BIOSS), University of Freiburg, Freiburg, Germany

    Wilfried Weber

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  1. Konrad Müller
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  2. Matias D Zurbriggen
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  3. Wilfried Weber
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Contributions

K.M. designed and performed the experiments, analyzed the data and wrote the paper; M.D.Z. and W.W. designed the experiments, analyzed the data and wrote the paper.

Corresponding author

Correspondence to Wilfried Weber.

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Competing interests

Patent applications have been filed (European Patent Office, World Intellectual Property Organization) by the Baden-Württemberg Stiftung covering this technology, for which K.M., M.D.Z. and W.W. are inventors.

Integrated supplementary information

Supplementary Figure 1 Oxidation of PCB in cell culture medium.

(A) Change in the absorbance spectrum. 15 μM PCB was added to cell culture medium and incubated at 37 °C, 5% CO2. At the indicated points in time after PCB addition, samples were removed and the absorbance spectrum between 300 nm and 700 nm was measured. (B) Color change of the PCB-supplemented medium-samples from (A).

Supplementary information

Oxidation of PCB in cell culture medium.

(A) Change in the absorbance spectrum. 15 μM PCB was added to cell culture medium and incubated at 37 °C, 5% CO2. At the indicated points in time after PCB addition, samples were removed and the absorbance spectrum between 300 nm and 700 nm was measured. (B) Color change of the PCB-supplemented medium-samples from (A). (PDF 240 kb)

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Müller, K., Zurbriggen, M. & Weber, W. Control of gene expression using a red- and far-red light–responsive bi-stable toggle switch. Nat Protoc 9, 622–632 (2014). https://doi.org/10.1038/nprot.2014.038

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