Live Imaging with Green Fluorescent Protein

  • Jim Haseloff
  • Emma-Louise Dormand
  • Andrea H. Brand
Part of the Methods in Molecular Biology™ book series (MIMB, volume 122)


If developmental biologists were given the chance to design the perfect cell-or tissue-specific marker, they would ensure that it had several properties. First, it would function in living animals, eliminating the need for fixation and dehydration and their associated artefacts. Second, it would permit each stage of development to be studied in a single, intact embryo. Third, it would function in all cell types and would reveal their morphology, making it simple to identify different cells without compromising their viability.


Green Fluorescent Protein Arabidopsis Seedling Green Fluorescent Protein Fluorescence Green Fluorescent Protein Fusion Vitelline Membrane 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


  1. 1.
    Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W., and Prasher, D. C. (1994) Green fluorescent protein as a marker for gene expression. Science 263, 802–805.PubMedCrossRefGoogle Scholar
  2. 2.
    Cubitt, A. B., Heim, R., Adams, S. R., Boyd, A. E., Gross, L. A., and Tsien, R. Y. (1995) Understanding, improving and using green fluorescent proteins. Trends Biochem. Sci. 20, 448–455.PubMedCrossRefGoogle Scholar
  3. 3.
    Prasher, D. C., Eckenrode, V. K., Ward, W. W., Prendergast, F. G., and Cormier, M. J. (1992) Primary structure of the aequorea victoria green fluorescent protein. Gene 111, 229–233.PubMedCrossRefGoogle Scholar
  4. 4.
    Prasher, D. C. (1995) Using GFP to see the light. Trends Genet. 11, 320–323.PubMedCrossRefGoogle Scholar
  5. 5.
    Gerdes, H. and Kaether, C. (1996) Green fluorescent protein: applications in cell biology. FEBS Lett. 389, 44–47.PubMedCrossRefGoogle Scholar
  6. 6.
    Davenport, D. and Nichol, J. A. C. (1955) Luminsecence in Hydromedusae. Proc. Royal Society Series B, 144, 399–411.CrossRefGoogle Scholar
  7. 7.
    Morin, J. G. and Hastings, J. W. (1971) Energy transfer in a bioluminescent system. J. Cellular Physiol. 77, 313–318.CrossRefGoogle Scholar
  8. 8.
    Morise, H., Shimomura, O., Johnson, F. H., and Winant, J. (1974) Intermolecular energy transfer in the bioluminescent system of Aequorea. Biochemistry 13, 2656–2662.PubMedCrossRefGoogle Scholar
  9. 9.
    Bokman, S. H. and Ward, W. W. (1981) Renaturation of Aequorea green fluorescent protein. Biochem. Biophys. Res. Comm. 101, 1372–1380.PubMedCrossRefGoogle Scholar
  10. 10.
    Ward, W. W., Cody, C. W., Hart, R. C., and Cormier, M. J. (1980) Spectrophotomeric identity of the energy transfer chromophores in Renilla and Aequorea green fluorescent proteins. Photochem. Photobiol. 31, 611–615.CrossRefGoogle Scholar
  11. 11.
    Cody, C. W., Prasher, D. C., Westler, W. M., Prendergast, F. H., and Ward, W. W. (1993) Chemical structure of the hexapeptide chromophore of the Aequorea green fluorescent protein. Biochemistry 32, 1212–1218.PubMedCrossRefGoogle Scholar
  12. 12.
    Heim, R., Prasher, D. C., and Tsien, R. Y. (1994) Wavelength mutations and posttranslational autoxidation of green fluorescent protein. Proc. Natl. Acad. Sci. USA 9, 12,501–12,504.CrossRefGoogle Scholar
  13. 13.
    Ormo, M., Cubitt, A. B., Kallio, K., Gross, L. A., Tsien, R. Y., and Remington, S. J. (1996) Crystal structure of the Aequorea victoria green fluorescent protein. Science 273, 1392–1395.PubMedCrossRefGoogle Scholar
  14. 14.
    Yang, F., Moss, L. G., and Phillips, G. N. (1996) The molecular structure of green fluorescent protein. Nature Biotechnol. 14, 1246–1251.CrossRefGoogle Scholar
  15. 15.
    Inouye, S. and Tsuji, F. I. (1994) Aequorea green fluorescent protein — expression of the gene and fluorescence characteristics of the recombinant protein. FEBS Lett. 341, 277–280.PubMedCrossRefGoogle Scholar
  16. 16.
    Wang, S. and Hazelrigg, T. (1994) Implications for bcd messenger RNA localization from spatial distribution of exu protein in Drosophila oogenesis. Nature 369, 400–403.PubMedCrossRefGoogle Scholar
  17. 17.
    Haseloff, J. and Amos, B. (1995) GFP in plants. Trends Genet. 11, 328,329.PubMedCrossRefGoogle Scholar
  18. 18.
    Rizzuto, R., Brini, M., De Giorgi, F., Rossi, R., Heim, R., Tsien, R. Y., and Pozzan, T. (1996) Double labelling of subcellular structures with organelle-targeted GFP mutants in vivo. Curr. Biol. 6, 183–188.PubMedCrossRefGoogle Scholar
  19. 19.
    Heim, R. and Tsien, R. Y. (1996) Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer. Curr. Biol. 6, 178–182.PubMedCrossRefGoogle Scholar
  20. 20.
    Haseloff, J., Siemering, D. R., Prasher, D. C., and Hodge, S. (1997) Removal of a cryptic intron and subcellular localisation of green fluorescent protein are required to mark transgenic Arabidopsis plants brightly. Proc. Natl. Acad. Sci. USA 94, 2122–2127.PubMedCrossRefGoogle Scholar
  21. 21.
    Reichel, C., Mathur, J., Eckes, P., Langenkemper, K., Reiss, B., Koncz, C., Schell, J., and Maas, C. (1996) Enhanced green fluorescence by the expression of an Aequorea victoria green fluorescent protein mutant in mono-and dicotyledonous plant cells. Proc. Natl. Acad. Sci. USA 93, 5888–5893.PubMedCrossRefGoogle Scholar
  22. 22.
    Chiu, W., Niwa, Y., Zeng, W., Hirano, T., Kobayashi, H., and Sheen, J. (1996) Engineered GFP as a vital reporter in plants. Curr. Biol. 6, 325–330.PubMedCrossRefGoogle Scholar
  23. 23.
    Haas, J., Park, E. C., and Seed, B. (1996) Codon usage limitation in the expression of HIV-1 envelope glycoprotein. Curr. Biol. 6, 315–324.PubMedCrossRefGoogle Scholar
  24. 24.
    Pang, S. Z., DeBoer, D. L., Wan, Y., Ye, G., Layton, J. G., Neher, M. K., Armstrong, C. L., Fry, J. E., Hinchee, M. A. W., and Fromm, M. E. (1996) An improved green fluorescent protein gene as a vital marker in plants. Plant Physiol. 112, 893–900.PubMedCrossRefGoogle Scholar
  25. 25.
    Siemering, K. R., Golbik, R., Sever, R., and Haseloff, J. (1996) Mutations that suppress the thermosensitivity of green fluorescent protein. Curr. Biol. 6, 1653–1663.PubMedCrossRefGoogle Scholar
  26. 26.
    Crameri, A., Whitehorn, E. A., Tate, E., and Stemmer, W. P. C. (1996) Improved green fluorescent protein by molecular evolution using DNA shuffling. Nature Biotechnol. 14, 315–319.CrossRefGoogle Scholar
  27. 27.
    Davis, S. J. and Viestra, R. D. (1998) Soluble, highly fluorescent variants of green fluorescent protein (GFP) for use in higher plants. Plant Mol. Biol. 36, 521–528.PubMedCrossRefGoogle Scholar
  28. 28.
    Kohler, R. H., Zipfel, W. R., Webb, W. W., and Hanson, M. R. (1997) The green fluorescent protein as a marker to visualize plant mitochondria in vivo. Plant J. 11, 613–621.PubMedCrossRefGoogle Scholar
  29. 29.
    Cormack, B. P., Valdivia, R. H., and Falkow, S. (1996) FACS-optimized mutants of the green fluorescent protein (GFP). Gene 173, 33–38.PubMedCrossRefGoogle Scholar
  30. 30.
    Zernicka-Goetz, M., Pines, J., Ryan, K., Siemering, K. R., Haseloff, J., Evans, M. J., and Gurdon, J. B. (1996) An indelible lineage marker for Xenopus using a mutated green fluorescent protein. Development 122, 3719–3724.PubMedGoogle Scholar
  31. 31.
    Zernicka-Goetz, M., Pines, J., Siemering, K. R., Haseloff, J., and Evans, M. J. (1997) Following cell fate in the living mouse embryo. Development 124, 1133–1137.PubMedGoogle Scholar
  32. 32.
    Schuldt, A., Adams, J. H. J., Davidson, C. M., Micklem, D. R., Haseloff, J., St Johnston, D., and Brand, A. H. (1998) Miranda mediates asymmetric protein and RNA localisation in the developing nervous system. Genes Develop. 12, 1847–1857.PubMedCrossRefGoogle Scholar
  33. 33.
    Delagrave, S., Hawtin, R. E., Silva, C. M., Yang, M. M., and Youvan, D. C. (1995) Red-shifted excitation mutants of the green fluorescent protein. Bio-Technology 13, 151–154.PubMedGoogle Scholar
  34. 34.
    Ehrig, T., OKane, D. J., and Predergast, F. G. (1995) Green fluorescent protein mutants with altered fluorescence excitation spectra. FEBS Lett. 367, 163–166.PubMedCrossRefGoogle Scholar
  35. 35.
    Heim, R., Cubitt, A. B. and Tsien, R. Y. (1995) Improved green fluorescence. Nature 373, 663,664.PubMedCrossRefGoogle Scholar
  36. 36.
    Stauber, R. H., Horie, K., Carney, P., Hudson, E. A., Tarasova, N. I., Gaitanaris, G. A., and Pavlakis, G. N. (1998) Development and applications of enhanced green fluorescent protein mutants. Biotechniques 24, 462–471.PubMedGoogle Scholar
  37. 37.
    Yang, T. T., Sinai, P., Green, G., Kitts, P. A., Chen, Y. T., Lybarger, L., Chervenak, R., Patterson, G. H., Piston, D. W., and Kain, S. R. (1998) Improved fluorescence and dual colour detection with enhanced blue and green variants of the green fluorescent protein. J. Biol. Chem. 273, 8212–8216.PubMedCrossRefGoogle Scholar
  38. 38.
    Elowitz, M. B., Surette, M. G., Wolf, P. E., Stock, J., and Leibler, S. (1997) Photoactivation turns green fluorescent protein red. Curr. Biol. 7, 809–812.PubMedCrossRefGoogle Scholar
  39. 39.
    Sawin, K. E. and Nurse, P. (1997) Photoactivation of green fluorescent protein. Curr. Biol. 7, 606,607.CrossRefGoogle Scholar
  40. 40.
    Ludin, B. and Matus, A. (1998) GFP illuminates the cytoskeleton. Trends Cell Biol. 8, 72–77.PubMedCrossRefGoogle Scholar
  41. 41.
    Butner, K. A. and Kirschner, M. W. (1991) Tau protein binds to microtubules through a flexible array of distributed weak sites. J. Cell Biol. 115, 717–730.PubMedCrossRefGoogle Scholar
  42. 42.
    Callahan, C. A. and Thomas, J. B. (1994) Tau-b-galactosidase, an axon-targeted fusion protein. Proc. Natl. Acad. Sci. USA 91, 5972–5976.PubMedCrossRefGoogle Scholar
  43. 43.
    Brand, A. (1995) GFP in Drosophila. Trends Genet. 11, 324,325.PubMedCrossRefGoogle Scholar
  44. 44.
    Micklem, D. R., Dasgupta, R., Elliott, H., Gergely, F., Davidson, C., Brand, A., Gonzalez-Reyes, A., and St Johnston, D. (1997) mago nashi is required for the polarisation of the oocyte and the formation of perpendicular axes in Drosophila. Curr. Biol. 7, 468–478.PubMedCrossRefGoogle Scholar
  45. 45.
    Dormand, E. L. and Brand, A. H. (1998) Runt determines cell fates in the Drosophila embryonic CNS. Development 125, 1659–1667.PubMedGoogle Scholar
  46. 46.
    Davis, I., Girdham, C. H., and OFarrell, P. H. (1995) A nuclear GFP that marks nuclei in living Drosophila embryos; maternal supply overcomes a delay in the appearance of zygotic fluorescence. Develop. Biol. 170, 726–729.PubMedCrossRefGoogle Scholar
  47. 47.
    Ogawa, H., Inouye, S., Tsuji, F. I., Yasuda, K., and Umesono, K. (1995) Localization, trafficking and temperature-dependence of the Aequorea green fluorescent protein in cultured vertebrate cells. Proc. Natl. Acad. Sci. USA 92, 11,899–11,903.PubMedCrossRefGoogle Scholar
  48. 48.
    Rizzuto, R., Brini, M., Pizzo, P., Murgia, M., and Pozzan, T. (1995) Chimeric green fluorescent protein as a tool for visualizing subcellular organelles in living cells. Curr. Biol. 5, 635–642.PubMedCrossRefGoogle Scholar
  49. 49.
    Shiga, Y., Tanakamatakatsu, M., and Hayashi, S. (1996) A nuclear GFP beta-galactosidase fusion protein as a marker for morphogenesis in living Drosophila. Dev. Growth Differ. 38, 99–106.CrossRefGoogle Scholar
  50. 50.
    Moriyoshi, K., Richards, L. J., Akazawa, C., OLeary, D. D. M., and Naanishi, S. (1996) LLabeling neural cells using adenoviral gene transfer of membrane-targeted GFP. Neuron 116, 255–260.CrossRefGoogle Scholar
  51. 51.
    Terasaki, M., Jaffe, L. A., Hunnicutt, G. R., and Hammer, J. A. (1996) Structural change of the endoplasmic reticulum during fertilization: evidence for loss of membrane continuity using the green fluorescent protein. Develop. Biol. 179, 320–328.PubMedCrossRefGoogle Scholar
  52. 52.
    Marshall, J., Molloy, R., Moss, G., Howe, J., and Hughes, T. (1996) The jellyfish green fluorescent protein: a new tool for studying ion channel expression and function. Neuron 14, 211–215.CrossRefGoogle Scholar
  53. 53.
    Kaether, C. and Gerdes, H. (1995) Visualization of protein transport along the secretory pathway using green fluorescent protein. FEBS Lett. 396, 267–271.CrossRefGoogle Scholar
  54. 54.
    Presley, J. F., Cole, N. B., Schroer, T. A., Hirschberg, K., Zaal, K. J. M., and Lippincott-Schwartz, J. (1997) ER-to-Golgi transport visualized in living cells. Nature 389, 81–85.PubMedCrossRefGoogle Scholar
  55. 55.
    Schackwitz, W. S., Inoue, T., and Thomas, J. H. (1996) Chemosensory neurons function in parallel to mediate a pheromone response in C. elegans. Neuron 17, 719–728.PubMedCrossRefGoogle Scholar
  56. 56.
    Valvekens, D., Van Montagu, M., and Van Lijsebettens, M. (1988) Agrobacterium tumefaciens-mediated transformation of Arabidopsis thaliana root explants by using kanamycin selection. Proc. Natl. Acad. Sci. USA 85, 5536–5540.PubMedCrossRefGoogle Scholar
  57. 57.
    Patel, N. H. (1994) Imaging neuronal subsets and other cell types in whole-mount Drosophila embryos and larvae using antibody probes, in Drosophila melanogaster: Practical Uses in Cell and Molecular Biology (Goldstein, L. S. B. and Fyrberg, E. A., eds.), Academic Press, San Diego, pp. 446–485.Google Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 1999

Authors and Affiliations

  • Jim Haseloff
    • 1
  • Emma-Louise Dormand
    • 1
  • Andrea H. Brand
    • 1
  1. 1.Wellcome/CRC InstituteUniversity of CambridgeUK

Personalised recommendations