Advertisement

Establishment of Human Cell Lines Lacking Mitochondrial DNA

  • Kazunari Hashiguchi
  • Qiu-Mei Zhang-Akiyama
Protocol
Part of the Methods in Molecular Biology™ book series (MIMB, volume 554)

Abstract

Mitochondria have their own genome, and mitochondrial DNA (mtDNA) encodes 2 ribosomal RNAs, 22 transfer RNAs, and 13 polypeptides that function in oxidative phosphorylation (OXPHOS). mtDNA mutations lead to dysfunction of OXPHOS, resulting in cell death and/or compromised cellular activity. Cell lines lacking mtDNA (termed ρ0 cells) are very effective tools for studying the consequences of mtDNA mutations. ρ0cell lines have been used widely to investigate relationships between mtDNA mutation, mitochondrial function, and a variety of cellular processes. In this chapter, we summarize the yeast and animal ρ0 cell lines that have been studied. We provide simple protocols for the generation of human ρ0 cells by exposure to ethidium bromide and PCR verification of their ρ0 status.

Key words

Mitochondria, mitochondrial DNA (mtDNA), ρ0 cells, oxidative phosphorylation (OXPHOS), uridine pyruvate, ethidium bromide, PCR 
This is a preview of subscription content, log in to check access.

References

  1. 1.
    Gillham, N.W. (1994) Organelle Genes and Genomes. Oxford University Press, New York, NY.Google Scholar
  2. 2.
    Hashiguchi, K., Bohr, V.A. and de Souza-Pinto, N.C. (2004) Oxidative stress and mitochondrial DNA repair: implications for NRTIs induced DNA damage. Mitochondrion, 4, 215–222.CrossRefPubMedGoogle Scholar
  3. 3.
    Kang, D. and Hamasaki, N. (2002) Maintenance of mitochondrial DNA integrity: repair and degradation. Curr Genet, 41, 311–322.CrossRefPubMedGoogle Scholar
  4. 4.
    Shadel, G.S. and Clayton, D.A. (1997) Mitochondrial DNA maintenance in vertebrates. Annu Rev Biochem, 66, 409–435.CrossRefPubMedGoogle Scholar
  5. 5.
    Desjardins, P., Frost, E. and Morais, R. (1985) Ethidium bromide-induced loss of mitochondrial DNA from primary chicken embryo fibroblasts. Mol Cell Biol, 5, 1163–1169.PubMedGoogle Scholar
  6. 6.
    King, M.P. and Attardi, G. (1989) Human cells lacking mtDNA: repopulation with exogenous mitochondria by complementation. Science, 246, 500–503.CrossRefPubMedGoogle Scholar
  7. 7.
    Morais, R., Gregoire, M., Jeannotte, L. and Gravel, D. (1980) Chick embryo cells rendered respiration-deficient by chloramphenicol and ethidium bromide are auxotrophic for pyrimidines. Biochem Biophys Res Commun, 94, 71–77.CrossRefPubMedGoogle Scholar
  8. 8.
    Chandel, N.S. and Schumacker, P.T. (1999) Cells depleted of mitochondrial DNA (rho0) yield insight into physiological mechanisms. FEBS Lett, 454, 173–176.CrossRefPubMedGoogle Scholar
  9. 9.
    Hashiguchi, K., Stuart, J.A., de Souza-Pinto, N.C. and Bohr, V.A. (2004) The C-terminal alphaO helix of human Ogg1 is essential for 8-oxoguanine DNA glycosylase activity: the mitochondrial beta-Ogg1 lacks this domain and does not have glycosylase activity. Nucleic Acids Res, 32, 5596–5608.CrossRefPubMedGoogle Scholar
  10. 10.
    Stuart, J.A., Hashiguchi, K., Wilson, D.M., 3rd, Copeland, W.C., Souza-Pinto, N.C. and Bohr, V.A. (2004) DNA base excision repair activities and pathway function in mitochondrial and cellular lysates from cells lacking mitochondrial DNA. Nucleic Acids Res, 32, 2181–2192.CrossRefPubMedGoogle Scholar
  11. 11.
    Stuart, J.A., Mayard, S., Hashiguchi, K., Souza-Pinto, N.C. and Bohr, V.A. (2005) Localization of mitochondrial DNA base excision repair to an inner membrane-associated particulate fraction. Nucleic Acids Res, 33, 3722–3732.CrossRefPubMedGoogle Scholar
  12. 12.
    Hayashi, J., Ohta, S., Kagawa, Y., Kondo, H., Kaneda, H., Yonekawa, H., Takai, D. and Miyabayashi, S. (1994) Nuclear but not mitochondrial genome involvement in human age-related mitochondrial dysfunction. Functional integrity of mitochondrial DNA from aged subjects. J Biol Chem, 269, 6878–6883.PubMedGoogle Scholar
  13. 13.
    Hayashi, J., Ohta, S., Kikuchi, A., Takemitsu, M., Goto, Y. and Nonaka, I. (1991) Introduction of disease-related mitochondrial DNA deletions into HeLa cells lacking mitochondrial DNA results in mitochondrial dysfunction. Proc Natl Acad Sci U S A, 88, 10614–10618.CrossRefPubMedGoogle Scholar
  14. 14.
    Inoue, K., Ito, S., Takai, D., Soejima, A., Shisa, H., LePecq, J.B., Segal-Bendirdjian, E., Kagawa, Y. and Hayashi, J.I. (1997) Isolation of mitochondrial DNA-less mouse cell lines and their application for trapping mouse synaptosomal mitochondrial DNA with deletion mutations. J Biol Chem, 272, 15510–15515.CrossRefPubMedGoogle Scholar
  15. 15.
    Inoue, K., Nakada, K., Ogura, A., Isobe, K., Goto, Y., Nonaka, I. and Hayashi, J.I. (2000) Generation of mice with mitochondrial dysfunction by introducing mouse mtDNA carrying a deletion into zygotes. Nat Genet, 26, 176–181.CrossRefPubMedGoogle Scholar
  16. 16.
    Nakada, K., Inoue, K., Ono, T., Isobe, K., Ogura, A., Goto, Y.I., Nonaka, I. and Hayashi, J.I. (2001) Inter-mitochondrial complementation: Mitochondria-specific system preventing mice from expression of disease phenotypes by mutant mtDNA. Nat Med, 7, 934–940.CrossRefPubMedGoogle Scholar
  17. 17.
    Desjardins, P., de Muys, J.M. and Morais, R. (1986) An established avian fibroblast cell line without mitochondrial DNA. Somat Cell Mol Genet, 12, 133–139.CrossRefPubMedGoogle Scholar
  18. 18.
    Porteous, W.K., James, A.M., Sheard, P.W., Porteous, C.M., Packer, M.A., Hyslop, S.J., Melton, J.V., Pang, C.Y., Wei, Y.H. and Murphy, M.P. (1998) Bioenergetic consequences of accumulating the common 4977-bp mitochondrial DNA deletion. Eur J Biochem, 257, 192–201.CrossRefPubMedGoogle Scholar
  19. 19.
    Kukat, A., Kukat, C., Brocher, J., Schafer, I., Krohne, G., Trounce, I.A., Villani, G. and Seibel, P. (2008) Generation of rho0 cells utilizing a mitochondrially targeted restriction endonuclease and comparative analyses. Nucleic Acids Res, 36, e44.CrossRefPubMedGoogle Scholar
  20. 20.
    Gamen, S., Anel, A., Montoya, J., Marzo, I., Pineiro, A. and Naval, J. (1995) mtDNA-depleted U937 cells are sensitive to TNF and Fas-mediated cytotoxicity. FEBS Lett, 376, 15–18.CrossRefPubMedGoogle Scholar
  21. 21.
    Miller, S.W., Trimmer, P.A., Parker, W.D., Jr. and Davis, R.E. (1996) Creation and characterization of mitochondrial DNA-depleted cell lines with “neuronal-like” properties. J Neurochem, 67, 1897–1907.CrossRefPubMedGoogle Scholar
  22. 22.
    Inoue, K., Takai, D., Hosaka, H., Ito, S., Shitara, H., Isobe, K., LePecq, J.B., Segal-Bendirdjian, E. and Hayashi, J. (1997) Isolation and characterization of mitochondrial DNA-less lines from various mammalian cell lines by application of an anticancer drug, ditercalinium. Biochem Biophys Res Commun, 239, 257–260.CrossRefPubMedGoogle Scholar
  23. 23.
    Goldring, E.S., Grossman, L.I., Krupnick, D., Cryer, D.R. and Marmur, J. (1970) The petite mutation in yeast. Loss of mitochondrial deoxyribonucleic acid during induction of petites with ethidium bromide. J Mol Biol, 52, 323–335.CrossRefPubMedGoogle Scholar
  24. 24.
    Attardi, G., King, M.P., Chomyn, A. and Loguercio-Polosa, P. (1991) Novel genetic and molecular approaches to the study of mitochondrial biogenesis and mitochondrial diseases in human cells, in Progress in Neuropathology, vol. 7 (Sato T. and DiMauro, S., ed.), Raven Press, New York, pp. 75–92.Google Scholar
  25. 25.
    Bodnar, A.G., Cooper, J.M., Holt, I.J., Leonard, J.V. and Schapira, A.H. (1993) Nuclear complementation restores mtDNA levels in cultured cells from a patient with mtDNA depletion. Am J Hum Genet, 53, 663–669.PubMedGoogle Scholar
  26. 26.
    Herst, P.M., Tan, A.S., Scarlett, D.J. and Berridge, M.V. (2004) Cell surface oxygen consumption by mitochondrial gene knockout cells. Biochim Biophys Acta, 1656, 79–87.CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Kazunari Hashiguchi
    • 1
  • Qiu-Mei Zhang-Akiyama
    • 1
  1. 1.Laboratory of Radiation Biology, Graduate School of ScienceKyoto UniversityKyotoJapan

Personalised recommendations

Cite protocol

Buy options