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.
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References
Gillham, N.W. (1994) Organelle Genes and Genomes. Oxford University Press, New York, NY.
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.
Kang, D. and Hamasaki, N. (2002) Maintenance of mitochondrial DNA integrity: repair and degradation. Curr Genet, 41, 311–322.
Shadel, G.S. and Clayton, D.A. (1997) Mitochondrial DNA maintenance in vertebrates. Annu Rev Biochem, 66, 409–435.
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.
King, M.P. and Attardi, G. (1989) Human cells lacking mtDNA: repopulation with exogenous mitochondria by complementation. Science, 246, 500–503.
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.
Chandel, N.S. and Schumacker, P.T. (1999) Cells depleted of mitochondrial DNA (rho0) yield insight into physiological mechanisms. FEBS Lett, 454, 173–176.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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Hashiguchi, K., Zhang-Akiyama, QM. (2009). Establishment of Human Cell Lines Lacking Mitochondrial DNA. In: Stuart, J.A. (eds) Mitochondrial DNA. Methods in Molecular Biology™, vol 554. Humana Press. https://doi.org/10.1007/978-1-59745-521-3_23
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DOI: https://doi.org/10.1007/978-1-59745-521-3_23
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