Journal of Applied Biological Chemistry

The Korean Society for Applied Biological Chemistry (한국응용생명화학회)
권호 표지
DOI QR코드

DOI QR Code

Rapid Isolation of Mitochondrial DNA-Depleted Mammalian Cells by Ethidium Bromide and Dideoxycytidine Treatments

  • Yoon, Young Geol (Department of Biomedical Science, Jungwon University) ;
  • Oh, Yoo Jin (Department of Anatomy and Cell Biology, Mitochondria Hub Regulation Center, Dong-A University) ;
  • Yoo, Young Hyun (Department of Anatomy and Cell Biology, Mitochondria Hub Regulation Center, Dong-A University)
  • Received : 2014.03.06
  • Accepted : 2014.04.07
  • Published : 2014.09.30
Download PDF
⟨ Previous Next ⟩

Abstract

Mitochondrial DNA (mtDNA)-depleted (${\rho}^0$) cells are often used as mtDNA recipients to study the interaction between the nucleus and mitochondria in mammalian cells. Therefore, it is crucial to obtain mtDNA-depleted cells with many different nuclear backgrounds for the study. Here, we demonstrate a rapid and reliable method to isolate mammalian mtDNA-depleted cells involving treatment with the antimitochondrial agents ethidium bromide (EtBr) and 2',3'-dideoxycytidine (ddC). After a short exposure to EtBr or ddC, followed by rapid clonal isolation, we were able to generate viable mtDNA-depleted cells from mouse and human cells and were able to successfully repopulate them with exogenous mitochondria from platelets isolated from mouse and human blood samples. These mtDNA-depleted cells can be used to characterize the nuclear mitochondrial interactions and to study mtDNA-associated defects in mammalian cells. Our method of isolating mtDNA-depleted cells is practical and applicable to a variety of cell types.

Keywords

References

  1. Chen CH and Cheng YC (1989) Delayed cytotoxicity and selective loss of mitochondrial DNA in cells treated with the anti-human immunodeficiency virus compound 2',3'-dideoxycytidine. J Biol Chem 264, 11934-7.
  2. Chomyn A (1996) Platelet-mediated transformation of human mitochondrial DNA-less cells. Methods Enzymol 264, 334-9. https://doi.org/10.1016/S0076-6879(96)64031-2
  3. Dolezal P, Likic V, Tachezy J, and Lithgow T (2006) Evolution of the molecular machines for protein import into mitochondria. Science 313, 314-8. https://doi.org/10.1126/science.1127895
  4. Dupont J, Schwaller MA, and Dodin G (1990) Ditercalinium, a nucleic acid binder, inhibits the respiratory chain of isolated mammalian mitochondria. Cancer Res 50, 7966-72.
  5. Gray MW, Burger G, and Lang BF (1999) Mitochondrial evolution. Science 283, 1476-81. https://doi.org/10.1126/science.283.5407.1476
  6. Habteyesus A, Nordenskjold A, Bohman C, and Eriksson S (1991) Deoxynucleoside phosphorylating enzymes in monkey and human tissues show great similarities, while mouse deoxycytidine kinase has a different substrate specificity. Biochem Pharmacol 42,1829-36. https://doi.org/10.1016/0006-2952(91)90522-7
  7. Inoue K, Takai D, Hosaka H, Ito S, Shitara H, Isobe K et al. (1997) Isolation and characterization of mitochondrial DNA-less lines from various mammalian cell lines by application of an anticancer drug, ditercalinium. Biochem Biophy Res Comm 239, 257-60. https://doi.org/10.1006/bbrc.1997.7446
  8. Kenyon L and Moraes CT (1997) Expanding the functional human mitochondrial DNA database by the establishment of primate xenomitochondrial cybrids. Proc Natl Acad Sci USA 94, 9131-5. https://doi.org/10.1073/pnas.94.17.9131
  9. King MP and Attardi G (1996) Isolation of human cell lines lacking mitochondrial DNA. Methods Enzymol 264, 304-13. https://doi.org/10.1016/S0076-6879(96)64029-4
  10. Kukat A, Kukat C, Brocher J, Schafer I, Krohne G, Trounce IA et al. (2008) Generation of $n^0$ cells utilizing a mitochodrially targeted restriction endonuclease and comparative analyses. Nucleic Acid Res 36, e44. https://doi.org/10.1093/nar/gkn124
  11. Martin JL, Brown CE, Matthews-Davis N, and Reardon JE (1994) Effects of antiviral nucleoside analogs on human DNA polymerases and mitochondrial DNA synthesis. Antimicrob Agents Chemother 38, 2743-9. https://doi.org/10.1128/AAC.38.12.2743
  12. Mitsuya H and Broder S (1986) Inhibition of the in vitro infectivity and cytopathic effect of human T-lymphotrophic virus type III/lymphadenopathy-associated virus (HTLV-III/LAV) by 2',3'-dideoxynucleosides. Proc Natl Acad Sci USA 83, 1911-5. https://doi.org/10.1073/pnas.83.6.1911
  13. Nelson I, Hanna MG, Wood NW, and Harding AE (1997) Depletion of mitochondrial DNA by ddC in untransformed human cell lines. Somat Cell Mol Genet 23, 287-90. https://doi.org/10.1007/BF02674419
  14. Rossi L, Brandi G, Schiavano GF, Chiarantini L, Albano A, and Magnani M (1992) In vitro and in vivo toxicity of 2',3'-dideoxycytidine in mice. Chem Biol Interact 85, 255-63. https://doi.org/10.1016/0009-2797(92)90066-T
  15. Rossi L, Serafini S, Schiavano GF, Casabianca A, Vallanti G, Chiarantini L et al. (1999) Metabolism, mitochondrial uptake and toxicity of 2',3'-dideoxycytidine. Biochem J 344, 915-20. https://doi.org/10.1042/0264-6021:3440915
  16. Smith CA (1977) Absence of ethidium bromide induced nicking and degradation of mitochondrial DNA in mouse L-cells. Nucleic Acids Res 4, 1419-26. https://doi.org/10.1093/nar/4.5.1419
  17. Smith CA, Jordan JM, and Vinograd J (1971) In vivo effects of intercalating drugs on the superhelix density of mitochondrial DNA isolated from human and mouse cells in culture. J Mol Biol 59, 255-72. https://doi.org/10.1016/0022-2836(71)90050-7
  18. Yarchoan R and Broder S (1987) Development of antiretroviral therapy for the acquired immunodeficiency syndrome and related disorders. A progress report. N Engl J Med 316, 557-64.
  19. Yoon YG, Koob MD, and Yoo YH (2010) Re-engineering the mitochondrial genomes in mammalian cells. Anat Cell Biol 43, 97-109. https://doi.org/10.5115/acb.2010.43.2.97

Cited by

  1. in granary vol.59, pp.2, 2016, https://doi.org/10.3839/jabc.2016.023
  2. in the granary vol.59, pp.3, 2016, https://doi.org/10.3839/jabc.2016.035