Abstract
Mycoplasma contamination of tissue culture cells easily evades detection and, thus, represents a continous threat to cell biologists. In cases where infected cell can not simply be replaced, attempts have to be made to eradicate mycoplasma from the tissue culture cells. A variety of anti-microbial agents have been shown to be toxic to mycoplasma strains; however, cell associated mycoplasmas are often protected from antibiotics at concentrations shown to be effectivein vitro. Antibiotic concentrations high enough to be lethal to cell asso|ciated mycoplasmas frequently are also detrimental to the host cells, while moderately increased antibiotic levels tolerated by the host cells often lead to only temporary growth suppression and/or to the emergence of mycoplasma strains resistant even to high concentrations of the antibiotic applied. Here, a genetic approach for the elimination of mycoplasma from tissue culture cells that overcomes these limitations is described. By expression of a selection marker conferring resistance to an otherwise toxic agent,Acholeplasma laidlawii infected BHK-21 cells used as the model system were enabled to temporarily tolerate antibiotic concentrations high enough to be lethal to cell associated mycoplasma while leaving the host cells unharmed. Upon successful mycoplasma eradication, cultivation of the cured host cells in the absence of the selective agent yielded revertant cell clones that had regained susceptibility to the toxic agent. Cessation of the selection marker expression was shown to result from the loss of the selection marker DNA, which is a consequence of the fact that the stable and permanent integration of foreign DNA in eucaryotic cell chromosomes is highly inefficient. Thus, the cells were cured from mycoplasma yet remained biochemically unaltered.
Similar content being viewed by others
References
Rottem, S. and M. E. Barile (1993) Beware of mycoplasmas.Tibtech 11: 143–151.
McGarrity, G. J., J. Sarama, and V. Vanaman (1985) Cell culture techniques.ASM News 51: 170–183.
Fleckenstein, E. and H. G. Drexler (1996) Elimination of mycoplasma contamination in cell cultures.Biochemica 1: 48–51.
Stanbridge, E. (1971) Mycoplasmas and cell cultures.Bacteriol. Rev. 35: 206–227.
McCarrity, G. J., V. Vanaman, and J. Sarama (1984) Cytogenetic effects of mycoplasmal infection of cell cultures: a review.In Vitro 20: 1–18.
MacPherson, I. (1966) Mycoplasmas in tissue culture.J. Cell. Sci. 1: 145–168.
Herderschee, D., A. C. Ruys, and G. R. van Rhijn (1963) Pleuropneumonia-like organisms in tissue cultures.Antonic van Leeuwenhock 29: 368–376.
Hay, R. J., M. L. Macy, and T. R. Chen (1989) Mycoplasma infection of cultured cells.Nature 339: 487–488.
Sprössig, M. and W. Witzleb (1968) Mycoplasma diseases of man. Custav Fischer Verlag, Jena, Germany.
Foy, H. M., Grayston J. T., Kenny, G. E., E. R. Alexander, and R. McMahan (1966) Epidemiology of mycoplasma pneu moniae infection in familie.JAMA 197: 859–866.
Uphoff, C. C., S. M. Cignac, and H. G. Drexler (1992) Mycoplasma contamination of various human leukemia cell lines.J. Immunol. Meth. 149: 43–53.
Wirth, M., M. Grashoff, L. Schuhmacher, and H. J. Hauser (1995) Mycoplasma detection by mycoplasma PCR ELISA.Biochemica 3: 33–35.
Barile, M. F. and R. T. Schimke (1963) A rapid chemical cultures.Proc. Soc. Exp. Biol. Med. 114: 676–679.
Broitman, N. L., C. M. Hoyd, C. A. Johnson, L. M. De La Maza, and E. M. Peterson (1992) Comparison of commerically available media for detection and isolation of ureaplasma urealyticum and mycoplasma hominis.J. Clin. Microbiol. 30: 1335–1337.
Vogelzang, A. A. and G. Compeer-Dekker (1969) Elimination of mycoplasma from various cell cultures.Antonic van Leeuwenhoek 35: 393–403.
Pollock, M. E. and G. E. Kenny (1963) Mammalian cell cultures contaminated with PPLO III. Elimiation of PPLO with specific antiserum.Proc. Soc. Exp. Biol. Med. 112: 176–181.
Harwick, H. J. and E. R. Fekety (1969) The antibiotic susceptibility of mycoplasma hominis.J Clin Path. 22: 483–485.
Friend, C., M. C. Patuleia, and J. B. Nelson (1966) Antibiotic effect of tylosin on a mycoplasma contaminant in a tissue culture leukemia cell line.Proc. Soc. Exp. Biol. Med. 121: 1009–1010.
Cori, G. B. and D. Y. Lee (1964) A method for eradication of mycoplasma infections in cell cultures.Proc. Soc. Exp. Biol. Med. 117: 918–921.
Balduzzi, P., and R. J. Charbonneau (1964) Decontamination of pleuropneumonia-like organism (PPLO) infected tissue cultures.Experientia 20: 651–652.
Perlman, D., S. B. Rahman, and J. B. Semar (1967) Antibiotic control of mycoplasma in tissue culture.Appl. Microbiol. 15: 82–85.
Schlokal, U., B. E. Fischer, S. Herlitschka, C. Antoine, A. Preininger, C. Mohr, M. Himmelspach, O. Kistner, F. G. Falkner, and F. Dorner (1996) Production of highly homogeneous and structurally intact recombinant von willebrand multimers by furin-mediated propeptide removalin vitro.Biotechnol. Appl. Biochem. 24: 257–267.
Corman, C., R. Padmanabhan, and B. H. Howard (1983) High efficiency DNA-mediated transformation of primate cells.Science 221: 551–553.
Palmiter, R. D., R. R. Behringer, C. J. Quaife, F. Maxwell, I. H. Maxwell, and R. L. Brinster (1987) Cell lineage ablation in transgenic mice by cell-specific expression of a toxin gene.Cell 50: 435–443.
Hämmerle, T., M. Himmelspach, F. Dorner, and F. G. Falkner (1997) A sensitive PCR assay system for the quantitation of viral genome equivalents: human immunodeficiency virus type 1 (HIV-1) and hepatitis B virus (HBV).Arch. Virol. 142: 1297–1306.
Larin, M. N., N. V. Saxby, C. M. Williamson, D. Buggey, and N. S. Kenright (1968)In vitro susceptibility of mycoplasma pneumoniae to tetracyclines.Antimicrob. Ag. Chemother. 1967, pp. 680–686.
Pollock, M. E., G. E. Kenny, and J. T. Syverton (1960) Isolation and elimination of pleuropneumonia-like organisms from mammalian cell cultures.Proc. Soc. Exp. Biol. Med. 105: 10–15.
Kaufman, R. J., P. C. Brown, and R. T. Schimke (1979) Amplified dihydrofolate reductase genes in unstably methotrexate-resistant cells are associated with double minute chromosomes.Proc. Natl. Acad. Sci. USA 76: 5669–5673.
Pallavicini, M. G., P. S. Deteresa, C. Rosette, W. Gray, and E. Wurm (1990) Effects of methotrexate on transfected DNA stability in mammalian cells.Mol. Cell. Biol. 10: 401–404.
Weidle, U., P. Buckel, and J. Wienberg (1988) Amplified expression constructs for human tissue-type plasminogen activator in chinese hamster ovary cells: in tability in the absence of selective pressure.Gene 66: 193–203.
Schweizer, H., W. Witzler, and T. H. Blumöhr (1970) Zur Kontamination von Gewebekulturen mit Myloplasmen.Archiv für die gesamte Virusforschung 30: 130–136.
Rahman, S. B., J. B. Semar, and D. Perlman (1967) Antibiotic resistance in mycoplasma isolates from tissue cultures.Appl. Microbiol. 15: 970.
Schlokat, U., M. Himmelspach, F. C. Falkner, and E. Dorner (1997) Permanent gene expression in mammalian cells: gene transfer and selection, pp. 33–63. In: Mammalian cell biotechnology for protein expression (eds. Hauser and Wagner), De Gruyter, Berlin.
Wirth, M., J. Bode, G. Zettlmeissl, and H. Hauser (1988) Isolation of overproducing recombinant mammalian cell lines by a fast and simple selection procedure.Gene 73: 419–426.
Stanbridge, E., M. Önen, F. T. Perkins, and L. Hayflick (1969) Karyological and morphological characteristics of human diploid cell strain WI-38 infected with mycoplasmas.Exp. Cell Res. 57: 39–410.
Cross, G. E., M. R. Goodman, and E. J. Shaw (1967) Detection and treatment of contaminating mycoplasmas in cell culture.Aust. J. Exp. Biol. Med. Sci. 45: 201–212.
Baseman, J. B., and J. G. Tully (1997) Mycophasmas: sophisticated, reemerging, and burdenend by their notoriety.Emerg. Inf. Dis. 3: 21–32.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Mohr, G., Preininger, A., Himmelspach, M. et al. Permanent mycoplasma removal from tissue culture cells: A genetic approach. Biotechnol. Bioprocess Eng. 5, 84–91 (2000). https://doi.org/10.1007/BF02931877
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF02931877