Toward genetic transformation of mitochondria in mammalian cells using a recoded drug-resistant selection marker

Young Geol Yoon; Michael Duane Koob

doi:10.1016/j.jgg.2011.03.005

Volume 38 Issue 4

Apr. 2011

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Article Navigation > Journal of Genetics and Genomics > 2011 > 38(4): 173-179

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Toward genetic transformation of mitochondria in mammalian cells using a recoded drug-resistant selection marker

doi: 10.1016/j.jgg.2011.03.005

Young Geol Yoon^a,
Michael Duane Koob^b

a
Mitochondria Hub Regulation Center and Department of Anatomy and Cell Biology, Dong-A University College of Medicine, 3-1 Dongdaesin-dong, Seo-gu, Busan 602-714, Republic of Korea
b
Department of Laboratory Medicine and Pathology and Institute of Human Genetics, University of Minnesota Medical School, 420 Delaware St. S.E., Minneapolis, MN 55455, USA

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Corresponding author: E-mail address: youngyoon97@gmail.com (Young Geol Yoon)
Received Date: 2010-12-10
Accepted Date: 2011-03-10
Rev Recd Date: 2011-03-07

Available Online: 2011-03-23

Publish Date: 2011-04-20

Abstract

Abstract

Due to technical difficulties, the genetic transformation of mitochondria in mammalian cells is still a challenge. In this report, we described our attempts to transform mammalian mitochondria with an engineered mitochondrial genome based on selection using a drug resistance gene. Because the standard drug-resistant neomycin phosphotransferase confers resistance to high concentrations of G418 when targeted to the mitochondria, we generated a recoded neomycin resistance gene that uses the mammalian mitochondrial genetic code to direct the synthesis of this protein in the mitochondria, but not in the nucleus (mitochondrial version). We also generated a universal version of the recoded neomycin resistance gene that allows synthesis of the drug-resistant proteins both in the mitochondria and nucleus. When we transfected these recoded neomycin resistance genes that were incorporated into the mouse mitochondrial genome clones into mouse tissue culture cells by electroporation, no DNA constructs were delivered into the mitochondria. We found that the universal version of the recoded neomycin resistance gene was expressed in the nucleus and thus conferred drug resistance to G418 selection, while the synthetic mitochondrial version of the gene produced no background drug-resistant cells from nuclear transformation. These recoded synthetic drug-resistant genes could be a useful tool for selecting mitochondrial genetic transformants as a precise technology for mitochondrial transformation is developed.
- Mitochondrial codon,
- Recode,
- Mitochondrial transformation,
- Selection marker,
- G418

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References(28)

References

[1]	Andersson, G.E., Kurland, C.G. An extreme codon preference strategy: codon reassignment Mol. Biol. Evol., 8 (1991),pp. 530-544
[2]	Barrientos, A., Barros, M.H., Valnot, I. et al. Cytochrome oxidase in health and disease Gene, 286 (2002),pp. 53-63
[3]	Barry, P., Pratt-Lowe, E., Luciw, P.A. Electroporation of T-cell and macrophage cell lines Bio-Rad Lab. Tech. Bull., 1349 (1989)
[4]	Boynton, J.E., Gillham, N.W. Methods Enzymol., 264 (1996),pp. 279-296
[5]	Butow, R.A., Henke, R.M., Moran, J.V. et al. Methods Enzymol., 264 (1996),pp. 265-278
[6]	Delorme, M.O., Henaut, A. Codon usage is imposed by the gene location in the transcription unit Curr. Genet., 20 (1991),pp. 353-358
[7]	DiMauro, S., Schon, E.A. Mitochondrial DNA mutations in human disease Am. J. Med. Genet., 106 (2001),pp. 18-26
[8]	Fox, T.D., Sanford, J.C., McMullin, T.W. Plasmids can stably transform yeast mitochondria lacking endogenous mtDNA Proc. Natl. Acad. Sci. USA, 85 (1988),pp. 7288-7292
[9]	Gilkerson, R.W., Schon, E.A., Hernandez, E. et al. Mitochondrial nucleoids maintain genetic autonomy but allow for functional complementation J. Cell Biol., 181 (2008),pp. 1117-1128
[10]	Irwin, M.H., Johnson, L.W., Pinkert, C.A. Isolation and microinjection of somatic cell-derived mitochondria and germline heteroplasmy in transmitochondrial mice Transgenic Res., 8 (1999),pp. 119-123
[11]	Johnston, S.A., Anziano, P.Q., Shark, K. et al. Mitochondrial transformation in yeast by bombardment with microprojectiles Science, 240 (1988),pp. 1538-1541
[12]	Kmiec, B., Woloszynska, M., Janska, H. Heteroplasmy as a common state of mitochondrial genetic information in plants and animals Curr. Genet., 50 (2006),pp. 149-159
[13]	Montoya, J., Ojala, D., Attardi, G. Distinctive features of the 5′-terminal sequences of the human mitochondrial mRNAs Nature, 290 (1981),pp. 465-470
[14]	Pecina, P., Houstkova, H., Hansikova, H. et al. Genetic defects of cytochrome c oxidase assembly Physiol. Res., 53 (2004),pp. S213-S223
[15]	Potter, H. Gene transfer by electroporation Bio-Rad Lab. Tech. Bull., 1346 (1988)
[16]	Rustenbeck, I., Eggers, G., Reiter, H. et al. Polyamine modulation of mitochondrial calcium transport. I. Stimulatory and inhibitory effects of aliphatic polyamines, aminoglucosides and other polyamine analogues on mitochondrial calcium uptake Biochem. Pharmacol., 56 (1998),pp. 977-985
[17]	Saraste, M. Science, 283 (1999),pp. 1488-1493
[18]	Schon, E.A., Gilkerson, R.W. Functional complementation of mitochondrial DNAs: mobilizing mitochondrial genetics against dysfunction Biochim. Biophys. Acta, 1800 (2010),pp. 245-249
[19]	Takeda, K., Tasai, M., Iwamoto, M. et al. Microinjection of cytoplasm or mitochondria derived from somatic cells affects parthenogenetic development of murine oocytes Biol. Reprod., 72 (2005),pp. 1397-1404
[20]	Trifunovic, A., Wredenberg, A., Falkenberg, M. et al. Premature ageing in mice expressing defective mitochondrial DNA polymerase Nature, 429 (2004),pp. 417-423
[21]	Wallace, D.C. Mitochondrial diseases in man and mouse Science, 283 (1999),pp. 1482-1488
[22]	Wallace, D.C. A mitochondrial paradigm for degenerative diseases and ageing Novartis Found. Symp., 235 (2001),pp. 247-263
[23]	Williams, B.A., Hirt, R.P., Lucocq, J.M. et al. Nature, 418 (2002),pp. 865-869
[24]	Yoon, Y.G., Koob, M.D. Nucleic Acids Res., 31 (2003),pp. 1407-1415
[25]	Yoon, Y.G., Koob, M.D. Transformation of isolated mammalian mitochondria by bacterial conjugation Nucleic Acids Res., 33 (2005),p. e139
[26]	Yoon, Y.G., Koob, M.D. Selection by drug resistance proteins located in the mitochondria of mammalian cells Mitochondrion, 8 (2008),pp. 345-351
[27]	Yoon, Y.G., Koob, M.D., Yoo, Y.H. Re-engineering the mitochondrial genomes in mammalian cells Anat. Cell Biol., 43 (2010),pp. 97-109
[28]	Yoon, Y.G., Yang, Y.W., Koob, M.D. Biotechnol. Lett., 31 (2009),pp. 1671-1676