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Delivery of mtZFNs into Early Mouse Embryos

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Zinc Finger Proteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1867))

Abstract

Mitochondrial diseases often result from mutations in the mitochondrial genome (mtDNA). In most cases, mutant mtDNA coexists with wild-type mtDNA, resulting in heteroplasmy. One potential future approach to treat heteroplasmic mtDNA diseases is the specific elimination of pathogenic mtDNA mutations, lowering the level of mutant mtDNA below pathogenic thresholds. Mitochondrially targeted zinc-finger nucleases (mtZFNs) have been demonstrated to specifically target and introduce double-strand breaks in mutant mtDNA, facilitating substantial shifts in heteroplasmy. One application of mtZFN technology, in the context of heteroplasmic mtDNA disease, is delivery into the heteroplasmic oocyte or early embryo to eliminate mutant mtDNA, preventing transmission of mitochondrial diseases through the germline. Here we describe a protocol for efficient production of mtZFN mRNA in vitro, and delivery of these into 0.5 dpc mouse embryos to elicit shifts of mtDNA heteroplasmy.

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Acknowledgments

This work was supported by the Medical Research Council, UK (MC_U105697135). We would like to thank Dr. Carlo Viscomi for help and stimulating discussions during the course of this work and the personnel at Phenomics animal care facilities for skillful technical assistance.

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Authors and Affiliations

  1. Mitochondrial Genetics Group, MRC Mitochondrial Biology Unit, University of Cambridge, Cambridge, UK

    Beverly J. McCann, Payam A. Gammage & Michal Minczuk

  2. Mammalian Embryo and Stem Cell Group, Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, UK

    Andy Cox & Magdalena Zernicka-Goetz

  3. Department of Mitochondrial Biology, Max Planck Institute for Biology of Ageing, Cologne, Germany

    James B. Stewart

Authors
  1. Beverly J. McCann
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  2. Andy Cox
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  3. Payam A. Gammage
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  4. James B. Stewart
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  5. Magdalena Zernicka-Goetz
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  6. Michal Minczuk
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Corresponding author

Correspondence to Michal Minczuk .

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Editors and Affiliations

  1. Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, Shanghai, China

    Jia Liu

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McCann, B.J., Cox, A., Gammage, P.A., Stewart, J.B., Zernicka-Goetz, M., Minczuk, M. (2018). Delivery of mtZFNs into Early Mouse Embryos. In: Liu, J. (eds) Zinc Finger Proteins. Methods in Molecular Biology, vol 1867. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8799-3_16

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  • DOI: https://doi.org/10.1007/978-1-4939-8799-3_16

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8798-6

  • Online ISBN: 978-1-4939-8799-3

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