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Design, Assembly, and Characterization of TALE-Based Transcriptional Activators and Repressors

  • Protocol
TALENs

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1338))

Abstract

Transcription activator-like effectors (TALEs) are modular DNA-binding proteins that can be fused to a variety of effector domains to regulate the epigenome. Nucleotide recognition by TALE monomers follows a simple cipher, making this a powerful and versatile method to activate or repress gene expression. Described here are methods to design, assemble, and test TALE transcription factors (TALE-TFs) for control of endogenous gene expression. In this protocol, TALE arrays are constructed by Golden Gate cloning and tested for activity by transfection and quantitative RT-PCR. These methods for engineering TALE-TFs are useful for studies in reverse genetics and genomics, synthetic biology, and gene therapy.

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Acknowledgements

This work was supported by a US National Institutes of Health (NIH) Director’s New Innovator Award (DP2OD008586), National Science Foundation (NSF) Faculty Early Career Development (CAREER) Award (CBET-1151035), NIH R01DA036865, NIH R21AR065956, NIH UH3TR000505, NIH P30AR066527, the Duke Coulter Translational Partnership, and an American Heart Association Scientist Development Grant (10SDG3060033). P.I.T. was supported by a National Science Foundation Graduate Research Fellowship and an American Heart Association Mid-Atlantic Affiliate Predoctoral Fellowship.

Author information

Authors and Affiliations

  1. Department of Biomedical Engineering, Duke University, Durham, NC, 27708, USA

    Pratiksha I. Thakore

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

    Charles A. Gersbach Ph.D.

  3. Center for Genomic and Computational Biology, Duke University, Durham, NC, 27708, USA

    Charles A. Gersbach Ph.D.

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

    Charles A. Gersbach Ph.D.

Authors
  1. Pratiksha I. Thakore
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  2. Charles A. Gersbach Ph.D.
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Corresponding author

Correspondence to Charles A. Gersbach Ph.D. .

Editor information

Editors and Affiliations

  1. Max-Delbrück-Center for Molecular Medicine, Berlin, Germany

    Ralf Kühn

  2. Institute of Developmental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health, Neuherberg, Germany

    Wolfgang Wurst

  3. Institute of Developmental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health, Neuherberg, Germany

    Benedikt Wefers

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Thakore, P.I., Gersbach, C.A. (2016). Design, Assembly, and Characterization of TALE-Based Transcriptional Activators and Repressors. In: Kühn, R., Wurst, W., Wefers, B. (eds) TALENs. Methods in Molecular Biology, vol 1338. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2932-0_7

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  • DOI: https://doi.org/10.1007/978-1-4939-2932-0_7

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2931-3

  • Online ISBN: 978-1-4939-2932-0

  • eBook Packages: Springer Protocols

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