Abstract
Treatment of mouse LMTK− cells with the toxic mitochondrial dye rhodamine 6G (R-6G) at 2.5 μg/ml for 7 days prevented cell growth while maintaining viability, with less than 10−6 cells recovering to form colonies. Pre-treatment of LMTK− cells with R-6G was followed by fusion with enucleated mouse 501–1 cells harboring a homoplasmic point mutation in the mitochondrial DNA (mtDNA) 16S rRNA gene conferring chloramphenicol resistance (CAPR). Cybrids and any surviving unfused LMTK− cells were selected in BrdU with or without CAP and their mtDNAs screened for the presence of the CAPR marker. Approximately 1 colony per 2×105 LMTK− cells appeared in the fusion plates selected both with and without CAP. Most clones investigated were confirmed to be cybrids by showing the presence of the generally homoplasmic CAPR mutation, whether or not CAP selection was used. Hence, R-6G pre-treatment permits construction of transmitochondrial cybrid cell lines carrying a variety of mtDNAs, without the need for ρo cell lines.
Literature Cited
Morais, R., Desjardins, P., Turmel, C., and Zinkewich-Peotti, K. (1987).In Vitro Cell Devel. Biol. 24:649–658.
King, M., and Attardi, G. (1989).Science 246:500–503.
Wallace, D.C., Bunn, C.L., and Eisenstadt, J.M. (1975).J. Cell Biol. 67:174–188.
Chomyn, A., Meola, G., Bresolin, N., Lai, S.T., Scarlato, G., and Attardi, G. (1991).Mol. Cell. Biol. 11:2236–2244.
Hayashi, J-I., Ohta, S., Kikuchi, A., Takemitsu, M., Goto, Y-I., and Nonaka, I. (1991).Proc. Natl. Acad. Sci. U.S.A. 88:10614–10618.
King, M., Koga, Y., Davidson, M., and Schon, E.A. (1992).Mol. Cell. Biol. 12:480–490.
Trounce, I., Neill, S., and Wallace, D.C. (1994)Proc. Natl. Acad. Sci. U.S.A. 91:8334–8338.
Ziegler, M.L., and Davidson, R.L. (1981).Somatic Cell Genet. 7:73–88.
Lampidis, T.J., Hasin, Y., Weiss, M.J., and Chen, L.B. (1985).Biomed. Pharmacother. 39:220–226.
Chen, L.B. (1988).Ann. Rev. Cell Biol. 4:155–181.
Mannella, C.A., and Wang, Q. (1989).Biochem. Biophys. Acta 981:363–366.
Johnson, L.V., Walsh, M.L., and Chen, L.B. (1980).Proc. Natl. Acad. Sci. U.S.A. 77:990–994.
Gear, A.R.L. (1974).J. Biol. Chem. 249:3628–3637.
Higuti, T., Niimi, S., Saito, R., Nakasima, S.N., Ohe, T., Tani, I., and Yoshimura, T. (1980).Biochem. Biophys. Acta 593:463–467.
Mai, M.S., and Allison, W.S. (1983).Arch. Biochem. Biophys. 221:467–476.
Wieker, H-J., Kuschmitz, D., and Hess, B. (1987).Biochem. Biophys. Acta 892:108–117.
Bullough, D.A., Ceccarelli, E.A., Roise, D., and Allison, W.S. (1989).Biochem. Biophys. Acta 975:377–383.
Kit, S., Dubbs, D.R., Piekarski, L.J., and Hsu, T.C. (1963).Exp. Cell. Res. 31:297–312.
Littlefield, J.W. (1964).Nature 203:1142–1144.
Bunn, C.L., Wallace, D.C., and Eisenstadt, J.M. (1974).Proc. Natl. Acad. Sci. U.S.A. 71:1681–1685.
Bunn, C.L., Wallace, D.C., and Eisenstadt, J.M. (1977).Somatic Cell Molec. Genet. 3:71–92.
Blanc, H., Wright, C.T., Bibb, M.J., Wallace, D.C., and Clayton, D.A. (1981).Proc. Natl. Acad. Sci. U.S.A. 78:3789–3793.
Bibb, M.J., Van Etten, R.A., Wright, C.T., Walberg, M.W., and Clayton, D.A. (1981).Cell 26:167–180.
Sandford, J.A., and Stubblefield, E. (1987).Somatic Cell Molec. Genet. 13:279–284.
Ziegler, M.L. (1982). InTechniques in Somatic Cell Genetics, (ed.) Shay, J.W. Plenum, New York, pp. 211–219.
Smith, R., Huston, M.M., Jenkins, R.N., Huston, D.P., and Rich, R.R. (1983).Nature 306:599–601.
Zinkewich-Peotti, K., Parent, M., and Morais, R. (1990).Cancer Res. 50:6675–6682.
Terasaki, M., Song, J., Wong, J.R., Weiss, M.J., and Chen, L.B. (1984).Cell 38:101–108.
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Trounce, I., Wallace, D.C. Production of transmitochondrial mouse cell lines by cybrid rescue of rhodamine-6G pre-treated L-cells. Somat Cell Mol Genet 22, 81–85 (1996). https://doi.org/10.1007/BF02374379
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DOI: https://doi.org/10.1007/BF02374379