On the Suitability of Red and Enhanced Green Fluorescent Protein (DsRed/EGFP) as Reporter Combination

  • Ch. Baumstark-Khan
  • Ch. E. Hellweg
  • G. Horneck
Conference paper

Abstract

Bioassays for toxic effects in mammals require the use of appropriate reporters in mammalian cell systems. Fluorescent Proteins of different colors offer the possibility to study different processes in the same living cell. Here the suitability of a separate and combined measurement of Enhanced Green Fluorescent Protein (EGFP) and the recently available Red Fluorescent Protein (DsRed) from Discosoma sp. was examined in order to create a combined bioassay for gene expression and cytotoxic effects. In stably transfected EGFP-expressing Chinese Hamster Ovary cells, the whole population shows strong green fluorescence, while stable transfection with pDsRed-N1 resulted in DsRed-expressing colonies in which red fluorescence was restraint to their center. DsRed-expressing cell lines with 90 % DsRed expression were isolated. EGFP and DsRed can be differentiated and quantified by microplate reader measurements (filter combinations 460/40+508/20 and 540/25+590/35) and FACS analysis (argon laser 488 nm). Thus, they are suitable for construction of a two-parameter-bioassay.

Keywords

Toxicity Codon Platinum Argon Iodide 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Gardner, H.S. Jr., L.M. Brennan, M.W. Toussaint, A.B. Rosenkranz, E.M. Boncavage-Hennesssey, M.J. Wolfe, Environ. Health Persp. 106Suppl. 6 (1998) 1299CrossRefGoogle Scholar
  2. 2.
    Heim, R. and Tsien, R.Y. (1996): Engineering green fluorescent protein for improved brightness, longer wavelength and fluorescence resonance energy transfer. Curr. Biol. 6, 178–182.PubMedCrossRefGoogle Scholar
  3. 3.
    Chalfie, M., Tu, Y., Euskirchen G., Ward, W. W., Prasher, D. C. (1994): Green Fluorescent Protein as a Marker for Gene Expression. Science 263, 802–805.PubMedCrossRefGoogle Scholar
  4. 4.
    Kain, S.R., Adams, M., Kondepudi, A., Yang, T.T., Ward, W.W., Kitts, P. (1995): The green fluorescent protein as a reporter of gene expression and protein localization. BioTechniques 19, 650–655.PubMedGoogle Scholar
  5. 5.
    Cheng, L., Fu, J., Tsukamoto, A., Hawley, R.G. (1996): Use of green fluorescent protein variants to monitor gene transfer and expression in mammalian cells. Nat. Biotechnol. 14, 606–609.PubMedCrossRefGoogle Scholar
  6. 6.
    Anderson, M.T., Tijoe, I.M., Parks, D.R., Herzenberg, L.A., Nolan, G.P., Lorincz, M.C. (1996): Simultaneous fluorescence-activated cell sorter analysis of two distinct transcriptional elements within a single cell using engineered green fluorescent proteins. Proc. Natl Acad. Sci. USA 93, 8508–8511.PubMedCrossRefGoogle Scholar
  7. 7.
    Tsien, R.Y. (1998): The Green Fluorescent Protein. In: C.C. Richardson, J.N. Abelson, C.R.H. Raetz, J.W. Thorner (eds.): Annual Review of Biochemistry. Annual Reviews, Palo Alto, CA, USA, Vol. 67, pp. 509–544.Google Scholar
  8. 8.
    Heim, R., Cubitt, A.B., Tsien, R.Y. (1995): Improved green fluorescence. Nature 373, 663–664.PubMedCrossRefGoogle Scholar
  9. 9.
    Cormack, B.P., Valdivia, R.H., Falkow, S. (1996): FACS-optimized mutants of the green fluorescent protein (GFP). Gene 173, 33–38.PubMedCrossRefGoogle Scholar
  10. 10.
    Yang, T.T., Cheng, L., Kain, S.R. (1996): Optimized codon usage and chromophore mutations provide enhanced sensitivity with the green fluorescent protein. Nucleic Acids Res. 24, 4592–4593.PubMedCrossRefGoogle Scholar
  11. 11.
    Zhang, G., Gurtu, V., Kain, S.R. (1996): An enhanced green fluorescent protein allows sensitive detection of gene transfer in mammalian cells. Biochem. Biophys. Res. Commun. 227, 707–711.PubMedCrossRefGoogle Scholar
  12. 12.
    Hunt, L., Jordan, M., De Jesus, M., Wurm, F.M. (1999): GFP-expressing mammalian cells for fast, sensitive, noninvasive cell growth assessment in a kinetic mode. Biotechnol. Bioeng. 65, 201–205.PubMedCrossRefGoogle Scholar
  13. 13.
    Sandman, K.E., Maria, S.S., Zlokarnik, G., Lippard, S.J. (1999): Rapid fluorescence-based reporter-gene assays to evaluate the cytotoxicity and antitumor drug potential of platinum complexes. Chem. Biol. 6, 541–551.PubMedCrossRefGoogle Scholar
  14. 14.
    Hellweg, C.E., Baumstark-Khan C, Rettberg, P., Horneck, G. (2001): The Suitability of Enhanced Green Fluorescent Protein (EGFP) as a Reporter Component for Bioassays. Anal. Chim. Acta 426, 175–184.CrossRefGoogle Scholar
  15. 15.
    Hellweg, C.E., Baumstark-Khan, C., Horneck, G. (2001): Enhanced Green Fluoescent Protein (EGFP) as reporter for biomonitoring of cytotoxic effects in mammalian cells. Anal. Chim. Acta 427, 191–199.CrossRefGoogle Scholar
  16. 16.
    Matz, M.V., Pradkov, A.F., Labas, Y.A., Savitsky, A.P., Zaraisky, A.G., Markelov, M.L., Lukyanov, S.A. (1999): Fluorescent proteins from nonbioluminescent Anthozoa species. Nat. Biotechnol. 17, 969–973.PubMedCrossRefGoogle Scholar
  17. 17.
    Li, X., Zhao, X., Fang, Y., Jiang, X., Duong, T., Fan, C., Huang, C.C., Kain, S.R. (1998): Generation of destabilized green fluorescent protein as a transcription reporter. J. Biol. Chem. 273, 34970–34975.PubMedCrossRefGoogle Scholar
  18. 18.
    Sambrook, J., Fritsch, E.F., Maniatis, T. (1989): Strategies for Cloning in Plasmid Vectors. In: J. Sambrook, E.F. Fritsch, T. Maniatis (eds.): Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, New York, pp. 1.53–1.73Google Scholar
  19. 19.
    Rechsteiner, M., Semin. Cell Biol. 1 (1990) 433Google Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2002

Authors and Affiliations

  • Ch. Baumstark-Khan
    • 1
  • Ch. E. Hellweg
    • 1
  • G. Horneck
    • 1
  1. 1.Radiation Biology Divison Institute of Aerospace MedicineDLR German Aerospace CenterKölnGermany

Personalised recommendations