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Light-Inducible Gene Regulation with Engineered Zinc Finger Proteins

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Photoswitching Proteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1148))

Abstract

The coupling of light-inducible protein–protein interactions with gene regulation systems has enabled the control of gene expression with light. In particular, heterodimer protein pairs from plants can be used to engineer a gene regulation system in mammalian cells that is reversible, repeatable, tunable, controllable in a spatiotemporal manner, and targetable to any DNA sequence. This system, Light-Inducible Transcription using Engineered Zinc finger proteins (LITEZ), is based on the blue light-induced interaction of GIGANTEA and the LOV domain of FKF1 that drives the localization of a transcriptional activator to the DNA-binding site of a highly customizable engineered zinc finger protein. This chapter provides methods for modifying LITEZ to target new DNA sequences, engineering a programmable LED array to illuminate cell cultures, and using the modified LITEZ system to achieve spatiotemporal control of transgene expression in mammalian cells.

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Acknowledgments

Ricardo E. Dolmetsch provided the original GI and LOV-VP16 plasmid constructs and Carlos F. Barbas, III provided ZFP constructs used to characterize the original LITEZ system. Graeme O’Connell and Chandra Tucker assisted in the design and programming of the custom LED array. This research was funded by an NSF CAREER Award (CBET-1151035), NIH Director’s New Innovator Award (1DP2-OD008586), NIH 1R01-DA036865, and a Ralph E. Powe Junior Faculty Enhancement Award from Oak Ridge Associated Universities. L.R.P. was supported by an NIH Biotechnology Training Grant to the Duke Center for Biomolecular and Tissue Engineering (T32GM008555) and the Duke Biomedical Engineering McChesney Fellowship.

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Authors and Affiliations

  1. Department of Biomedical Engineering, Duke University, Room 136 Hudson Hall, 90281, Durham, NC, 27708-0281, USA

    Lauren R. Polstein & Charles A. Gersbach Ph.D.

  2. Institute for Genome Sciences and Policy, Duke University, Durham, NC, 27708, USA

    Charles A. Gersbach Ph.D.

  3. Department of Orthopaedic Surgery, Duke University Medical Center, Durham, NC, 27710, USA

    Charles A. Gersbach Ph.D.

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  1. Lauren R. Polstein
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  2. Charles A. Gersbach Ph.D.
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Correspondence to Charles A. Gersbach Ph.D. .

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Editors and Affiliations

  1. Institute of Anatomy and Cell Biology, University of Heidelberg, Heidelberg, Germany

    Sidney Cambridge

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Polstein, L.R., Gersbach, C.A. (2014). Light-Inducible Gene Regulation with Engineered Zinc Finger Proteins. In: Cambridge, S. (eds) Photoswitching Proteins. Methods in Molecular Biology, vol 1148. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0470-9_7

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  • DOI: https://doi.org/10.1007/978-1-4939-0470-9_7

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-0469-3

  • Online ISBN: 978-1-4939-0470-9

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