Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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

Subjects

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.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it

$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

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.

References

  1. Klug, A. The discovery of zinc fingers and their development for practical applications in gene regulation. Proc. Japan Acad. 81, 87–102 (2005).

    Article  CAS  Google Scholar 

  2. Papworth, M., Kolasinska, P. & Minczuk, M. Designer zinc-finger proteins and their applications. Gene 366, 27–38 (2006).

    Article  CAS  PubMed  Google Scholar 

  3. Jamieson, A.C., Miller, J.C. & Pabo, C.O. Drug discovery with engineered zinc-finger proteins. Nat. Rev. Drug Discov. 2, 361–368 (2003).

    Article  CAS  PubMed  Google Scholar 

  4. Santiago, Y. et al. Targeted gene knockout in mammalian cells by using engineered zinc-finger nucleases. Proc. Natl. Acad. Sci. USA 105, 5809–5814 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Urnov, F.D. et al. Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature 435, 646–651 (2005).

    Article  CAS  PubMed  Google Scholar 

  6. Moehle, E.A. et al. Targeted gene addition into a specified location in the human genome using designed zinc finger nucleases. Proc. Natl. Acad. Sci. USA 104, 3055–3060 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Geurts, A.M. et al. Knockout rats via embryo microinjection of zinc-finger nucleases. Science 325, 433 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Doyon, Y. et al. Heritable targeted gene disruption in zebrafish using designed zinc-finger nucleases. Nat. Biotechnol. 26, 702–708 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Meng, X., Noyes, M.B., Zhu, L.J., Lawson, N.D. & Wolfe, S.A. Targeted gene inactivation in zebrafish using engineered zinc-finger nucleases. Nat. Biotechnol. 26, 695–701 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Foley, J.E. et al. Rapid mutation of endogenous zebrafish genes using zinc finger nucleases made by Oligomerized Pool ENgineering (OPEN). PLoS One 4, e4348 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  11. Kim, Y.G., Cha, J. & Chandrasegaran, S. Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain. Proc. Natl. Acad. Sci. USA 93, 1156–1160 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Kandavelou, K. et al. Targeted manipulation of mammalian genomes using designed zinc finger nucleases. Biochem. Biophys. Res. Commun. 388, 56–61 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Taylor, R.W. & Turnbull, D.M. Mitochondrial DNA mutations in human disease. Nat. Rev. Genet. 6, 389–402 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Schapira, A.H. Mitochondrial disease. Lancet 368, 70–82 (2006).

    Article  CAS  PubMed  Google Scholar 

  15. Minczuk, M., Papworth, M.A., Kolasinska, P., Murphy, M.P. & Klug, A. Sequence-specific modification of mitochondrial DNA using a chimeric zinc finger methylase. Proc. Natl. Acad. Sci. USA 103, 19689–19694 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Taylor, R.W., Chinnery, P.F., Turnbull, D.M. & Lightowlers, R.N. Selective inhibition of mutant human mitochondrial DNA replication in vitro by peptide nucleic acids. Nat. Genet. 15, 212–215 (1997).

    Article  CAS  PubMed  Google Scholar 

  17. Tanaka, M. et al. Gene therapy for mitochondrial disease by delivering restriction endonuclease SmaI into mitochondria. J. Biomed. Sci. 9, 534–541 (2002).

    CAS  PubMed  Google Scholar 

  18. Alexeyev, M.F. et al. Selective elimination of mutant mitochondrial genomes as therapeutic strategy for the treatment of NARP and MILS syndromes. Gene Ther. 15, 516–523 (2008).

    Article  CAS  PubMed  Google Scholar 

  19. Minczuk, M., Papworth, M.A., Miller, J.C., Murphy, M.P. & Klug, A. Development of a single-chain, quasi-dimeric zinc-finger nuclease for the selective degradation of mutated human mitochondrial DNA. Nucleic Acids Res. 36, 3926–3938 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Papworth, M. et al. Inhibition of herpes simplex virus 1 gene expression by designer zinc-finger transcription factors. Proc. Natl. Acad. Sci. USA 100, 1621–1626 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Cornu, T.I. et al. DNA-binding specificity is a major determinant of the activity and toxicity of zinc-finger nucleases. Mol. Ther. 16, 352–358 (2008).

    Article  CAS  PubMed  Google Scholar 

  22. Carroll, D., Morton, J.J., Beumer, K.J. & Segal, D.J. Design, construction and in vitro testing of zinc finger nucleases. Nat. Protoc. 1, 1329–1341 (2006).

    Article  CAS  PubMed  Google Scholar 

  23. Wright, D.A. et al. Standardized reagents and protocols for engineering zinc finger nucleases by modular assembly. Nat. Protoc. 1, 1637–1652 (2006).

    Article  PubMed  Google Scholar 

  24. Sander, J.D., Zaback, P., Joung, J.K., Voytas, D.F. & Dobbs, D. An affinity-based scoring scheme for predicting DNA-binding activities of modularly assembled zinc-finger proteins. Nucleic Acids Res. 37, 506–515 (2009).

    Article  CAS  PubMed  Google Scholar 

  25. Fu, F. et al. Zinc Finger Database (ZiFDB): a repository for information on C2H2 zinc fingers and engineered zinc-finger arrays. Nucleic Acids Res. 37, D279–D283 (2009).

    Article  CAS  PubMed  Google Scholar 

  26. Maeder, M.L. et al. Rapid 'open-source' engineering of customized zinc-finger nucleases for highly efficient gene modification. Mol. Cell 31, 294–301 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Sander, J.D., Zaback, P., Joung, J.K., Voytas, D.F. & Dobbs, D. Zinc Finger Targeter (ZiFiT): an engineered zinc finger/target site design tool. Nucleic Acids Res. 35, W599–W605 (2007).

    Article  PubMed  PubMed Central  Google Scholar 

  28. Pfanner, N. & Geissler, A. Versatility of the mitochondrial protein import machinery. Nat. Rev. Mol. Cell Biol. 2, 339–349 (2001).

    Article  CAS  PubMed  Google Scholar 

  29. Fernandez-Martinez, J. et al. Overlap of nuclear localisation signal and specific DNA-binding residues within the zinc finger domain of PacC. J. Mol. Biol. 334, 667–684 (2003).

    Article  CAS  PubMed  Google Scholar 

  30. Matheny, C., Day, M.L. & Milbrandt, J. The nuclear localization signal of NGFI-A is located within the zinc finger DNA binding domain. J. Biol. Chem. 269, 8176–8181 (1994).

    CAS  PubMed  Google Scholar 

  31. Gaines, G.L. III . In organello RNA synthesis system from HeLa cells. Methods Enzymol. 264, 43–49 (1996).

    Article  CAS  PubMed  Google Scholar 

  32. Leister, D. & Herrmann, J.M. Mitochondria Practical Protocols (Humana Press, Totowa, 2007).

  33. Copeland, W.C. Mitochondrial DNA (Humana Press, Totowa, 2002).

  34. Hartmann, C.M., Gehring, H. & Christen, P. The mature form of imported mitochondrial proteins undergoes conformational changes upon binding to isolated mitochondria. Eur. J. Biochem. 218, 905–910 (1993).

    Article  CAS  PubMed  Google Scholar 

  35. Taylor, S.W. et al. Characterization of the human heart mitochondrial proteome. Nat. Biotechnol. 21, 281–286 (2003).

    Article  CAS  PubMed  Google Scholar 

  36. D'Errico, I., Dinardo, M.M., Capozzi, O., De Virgilio, C. & Gadaleta, G. History of the Tfam gene in primates. Gene 362, 125–132 (2005).

    Article  CAS  PubMed  Google Scholar 

Download references

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.

Author information

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

Authors
  1. Michal Minczuk
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Paulina Kolasinska-Zwierz
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Michael P Murphy
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Monika A Papworth
    View author publications

    You can also search for this author in PubMed Google Scholar

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.

Corresponding author

Correspondence to Michal Minczuk.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

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)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2009.245

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research