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Construction and testing of engineered zinc-finger proteins for sequence-specific modification of mtDNA

Nature Protocols volume 5, pages 342–356 (2010)Cite this article

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Abstract

Engineered zinc-finger proteins (ZFPs) are hybrid proteins developed to direct various effector domains (EDs) of choice to predetermined DNA sequences. They are used to alter gene expression and to modify DNA in a sequence-specific manner in vivo and in vitro. Until now, ZFPs have mostly been used to target DNA sites in nuclear genomes. This protocol describes how to adapt engineered ZFP technology to specifically modify the mammalian mitochondrial genome. The first step describes how to construct mitochondrially targeted ZFPs (mtZFPs) so that they are efficiently imported into mammalian mitochondria. In the second step, methods to test the basic properties of mtZFPs in vitro are described. Finally, we outline how the mtZFPs can be transiently transfected into mammalian cells and their mitochondrial import tested by both immunofluorescence and biochemical methods. The protocol can be completed within a week, although time-consuming DNA cloning steps may extend this.

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Figure 1: Current and potential application of zinc-finger proteins (ZFPs) for targeting and modifying the mitochondrial genome.
Figure 2: Method of preparing electrophoretic mobility shift assay (EMSA) reactions.
Figure 3: Example of a typical electrophoretic mobility shift assay (EMSA) experiment with mitochondrially targeted zinc-finger protein (mtZFP).
Figure 4: Example of an in vitro assay of mitochondrially targeted zinc-finger nuclease (mtZFN) activity.
Figure 5: Example of mitochondrially localized mitochondrially targeted zinc-finger protein (mtZFP) as analyzed by immunofluorescence.
Figure 6: Example of intra-cellular localization of mitochondrially targeted zinc-finger protein (mtZFP) analyzed by cell fractionation.

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Acknowledgements

This work was supported by the Medical Research Council, UK and the Federation of European Biochemical Societies Long-Term Fellowship (M.M.) and Federation of European Biochemical Societies Short-Term Fellowship (P.K.-Z.). We thank M. Moore, Mark I. and Y. Choo for their contribution in developing the presented methods. We are grateful to Sangamo Biosciences for providing us with many of the mtDNA-specific zinc-finger peptides that were used in the development of the presented protocols. We also thank Joanna Rorbach for her help on the manuscript.

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Author notes

  1. Paulina Kolasinska-Zwierz & Monika A Papworth

    Present address: Present addresses: The Gurdon Institute and Department of Genetics, University of Cambridge, Cambridge, UK (P.K.-Z.); MedImmune, Cambridge, UK (M.A.P.).,

Authors and Affiliations

  1. Mitochondrial Biology Unit, MRC, Cambridge, UK

    Michal Minczuk & Michael P Murphy

  2. Laboratory of Molecular Biology, MRC, Cambridge, UK

    Paulina Kolasinska-Zwierz & Monika A Papworth

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  1. Michal Minczuk
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Contributions

M.M., M.A.P and M.P.M. designed the research; M.M., M.A.P. and P.K.-Z. carried out the experiments; M.M., M.A.P., P.K.-Z. and M.P.M. analyzed data; and M.M., M.A.P. and M.P.M. wrote the paper.

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Correspondence to Michal Minczuk.

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Supplementary information

Supplementary Figure 1

(a) DNA cloning strategy as described in Steps 1-5 of the main text. The MTS from the File subunit of the human mitochondrial ATP synthase is indicated in red, the NES from the NS2 protein of MVM is indicated in yellow. Exemplary ZFP21 (Minczuk et al., 2006) cloned between the MTS and NES is indicated in cyan and the HA epitope tag is in green. (b) DNA and protein sequence of the mtZFP insert constructed according to the description of Steps 1-5 and illustrated in (a). (PDF 58 kb)

Supplementary Fig. 2

An illustration of a small-scale “homogeniser” constructed by inserting of a plunger of 1 ml syringe (with a rubber tip) into a standard 1.5 ml eppendorf tube. By moving the plunger up and down suction force is created that efficiently disrupts cells. We have tested other small-scale homogenisers e.g. 0.1 ml homogeniser mortar (Fisher Scientific, cat. no. FB56673) or motor driven pellet pestle (Cordless motor, SIGMA cat. no. Z359971 with 1.5 polypropylene pellet pestle (SIGMA cat. no. Z359947), however, our “home-made” plunger-eppendorf tube homogeniser gave the most efficient cell disruption in the presented mitochondria isolation protocol. (PDF 33 kb)

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Minczuk, M., Kolasinska-Zwierz, P., Murphy, M. et al. Construction and testing of engineered zinc-finger proteins for sequence-specific modification of mtDNA. Nat Protoc 5, 342–356 (2010). https://doi.org/10.1038/nprot.2009.245

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