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Purified Protein Delivery to Activate an Epigenetically Silenced Allele in Mouse Brain

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Book cover Epigenome Editing

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1767))

Abstract

The ability to activate or repress specific genes in the brain could have a tremendous impact for understanding and treating neurological disorders. Artificial transcription factors based on zinc finger, TALE, and CRISPR/Cas9 programmable DNA-binding platforms have been widely used to regulate the expression of specific genes in cultured cells, but their delivery into the brain represents a critical challenge to apply such tools in live animals. In previous work, we developed a purified, zinc finger-based artificial transcription factor that could be injected systemically, cross the blood-brain barrier, and alter expression of a specific gene in the brain of an adult mouse model of Angelman syndrome. Importantly, our mode of delivery produced widespread distribution throughout the brain. Here we describe our most current methods for the production and purification of the factor, dosage optimization, and use of live animal fluorescence imaging to visualize the kinetics of distribution.

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Acknowledgment

We thank Alexa Adams, Jennifer Trang Nguyen, Victoria Le, Anvita Komarla, Joanna Watterson, Andy Tran, Joshua Mandella, Ruth Le, and Michelle McAllister for their assistance with the experiments. We thank Enoch Baldwin and Sarah Lockwood for expert advice and discussions in developing these methods. Imaging work was performed at the Center for Molecular and Genomic Imaging (CMGI), University of California, Davis. We would like to acknowledge Michelle Connell for her help with Maestro 2 imaging. This work was supported by the NIH (NS071028), the Angelman Syndrome Foundation, and the Foundation for Angelman Syndrome Therapeutics. B.J.B. was also funded by an NSF fellowship (0707429) and a grant to UC Davis from the Howard Hughes Medical Institute through the Med into Grad Initiative (56005706) and a CTSC pilot study (TR000002). Maestro imaging was supported by a CMGI pilot grant.

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Authors and Affiliations

  1. Genome Center and Department of Biochemistry and Molecular Medicine, University of California, Davis, Davis, CA, USA

    Benjamin Pyles, Barbara J. Bailus, Henriette O’Geen & David J. Segal

  2. The Buck Institute for Research on Aging, Novato, CA, USA

    Barbara J. Bailus

Authors
  1. Benjamin Pyles
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  2. Barbara J. Bailus
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  3. Henriette O’Geen
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  4. David J. Segal
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Corresponding author

Correspondence to David J. Segal .

Editor information

Editors and Affiliations

  1. Department of Biochemistry, Institute of Biochemistry and Technical Biochemistry, Stuttgart, Germany

    Albert Jeltsch

  2. Department of Pathology and Medical Biology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands

    Marianne G. Rots

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Pyles, B., Bailus, B.J., O’Geen, H., Segal, D.J. (2018). Purified Protein Delivery to Activate an Epigenetically Silenced Allele in Mouse Brain. In: Jeltsch, A., Rots, M. (eds) Epigenome Editing. Methods in Molecular Biology, vol 1767. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7774-1_12

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  • DOI: https://doi.org/10.1007/978-1-4939-7774-1_12

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7773-4

  • Online ISBN: 978-1-4939-7774-1

  • eBook Packages: Springer Protocols

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