Cell Lineage Analysis

Applications of Green Fluorescent Protein
  • Magdalena Zernicka-Goetz
  • Jonathon Pines
Part of the Methods in Molecular Biology™ book series (MIMB, volume 135)


The ideal cell lineage marker is one that can be visualized in living tissues without perturbing development. Exogenously applied dyes are very useful, but in many instances there would be advantages to an endogenously expressed marker. Green fluorescent protein (GFP) appears to have the potential to be ideal for this purpose, and with its advent, lineage analysis is entering a new phase in which cells can now be followed in real time in living embryos. GFP is a 27-kDa protein found in the jellyfish Aequorea victoria that absorbs blue light and emits green light. It has the valuable property that the formation of the chromophore is an autocatalytic event that requires no cofactors. Furthermore, the cDNA that encodes GFP has been cloned, and thus GFP can be expressed and will become fluorescent in any organism or cell type. In this chapter, we describe the properties of GFP and the different mutants available to study cell lineages in different organisms. We will mainly concentrate on their application to the analysis of cell lineages in frogs and mice.


Green Fluorescent Protein Codon Usage Yellow Fluorescent Protein Green Fluorescent Protein Fluorescence Green Fluorescent Protein Signal 
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.
    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
  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.
    Heim, R., Cubitt, A. B., and Tsien, R. Y. (1995) Improved green fluorescence. Nature 373, 663–664.PubMedCrossRefGoogle Scholar
  4. 4.
    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
  5. 5.
    Ormö, 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
  6. 6.
    Zolotukhin, S., Potter, M., Hauswirth, W. W., Guy, J., and Muzyczka, N. (1996) A “humanized” green fluorescent protein cDNA adapted for high-level expression in mammalian cells. J. Virol. 70, 4646–4654.PubMedGoogle Scholar
  7. 7.
    Haseloff, J., Siemering, K. 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 Arabydopsis plants brightly. Proc. Natl. Acad. Sci. USA 94, 2122–2127.PubMedCrossRefGoogle Scholar
  8. 8.
    Zernicka-Goetz, M., Pines, J., Ryan, K., Siemering, K. R., Haseloff, J., and Gurdon, J. B. (1996) An indelible lineage marker for Xenopus using a mutated green fluorescent protein. Development 122, 3719–3724.PubMedGoogle Scholar
  9. 9.
    Zernicka-Goetz, M., Pines, J., Dixon, J., Hunter, S., Siemering, K. R., Haseloff, J., and Evans, M. J. (1997) Following cell fate in the living mouse embryo. Development 124, 1133–1137.PubMedGoogle Scholar
  10. 10.
    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
  11. 11.
    Chalfie, M., Tu, Y., Euskirchen, G., Ward, W., and Prasher, D. (1994) Green fluorescent protein as a marker for gene expression. Science 263, 802–805.PubMedCrossRefGoogle Scholar
  12. 12.
    Gerisch, G., Albrecht, R., Heizer, C., Hodgkinson, S., and Maniak, M. (1995) Chemoattractant controlled accumulation of coronin at the leading edge of Dictostilium cells monitored using green fluorescent protein-coronin fusion protein. Curr. Biol. 5, 1280–1285.PubMedCrossRefGoogle Scholar
  13. 13.
    Moores, S. L., Sabry, J. H., and Spudich, J. A. (1996) Myosin dynamics in live Dictyostelium cells. Proc. Natl. Acad. Sci. USA 93, 443–446.PubMedCrossRefGoogle Scholar
  14. 14.
    Nabeshima, K., Kurooka, H., Takeuchi, M., Kinoshita, K., Nakaseko, Y., and Yanagida, M. (1995) p93dis1, which is required for sister chromatid separation, is a novel microtubule and spindle pole body-associating protein phosphorylated at the Cdc2 target sites. Genes Dev. 9, 1572–1585.PubMedCrossRefGoogle Scholar
  15. 15.
    Doyle, T. and Botstein, D. (1996) Movement of yeast cortical actin cytoskeleton visualized in vivo. Proc. Natl. Acad. Sci. USA 93, 3886–3891.PubMedCrossRefGoogle Scholar
  16. 16.
    Davis, I., Girdham, C. H., and O’Farrell, P. H. (1995) A nuclear GFP that marks nuclei in living Drosophila embryos; maternal supply overcomes a delay in the appearance of zygotic fluorescence. Dev. Biol. 170, 726–729.PubMedCrossRefGoogle Scholar
  17. 17.
    Kerrebrock, A. W., Moore, D. P., Wu, J. S., and Orr Weaver, T. L. (1995) Mei-S332, a Drosophila protein required for sister-chromatid cohesion, can localize to meiotic centromere regions. Cell 83, 247–256.Google Scholar
  18. 18.
    Amsterdam, A., Lin, S., Moss, L. G., and Hopkins, N. (1996) Requirements for green fluorescent protein detection in transgenic zebrafish embryos. Gene 173, 99–103.PubMedCrossRefGoogle Scholar
  19. 19.
    Tannahill, D., Bray, S., and Harris, W. A. (1995) A Drosophila E(spl) gene is “neurogenic” in Xenopus: A green fluorescent protein study. Dev. Biol. 168, 694–697.PubMedCrossRefGoogle Scholar
  20. 20.
    Whittingham, D. G. (1971) Culture of mouse ova. J. Reprod. Fert. 14(suppl), 7–21.Google Scholar
  21. 21.
    Beddington, R. and Lawson, K. A. (1990) Clonal analysis of cell lineages, in Post-Implantation Mammalian Embryos: A Practical Approach, (Copp, A. J. and Cockcroft, D. L., eds.), IRL, Oxford, UK.Google Scholar
  22. 22.
    Lawitts, J. A. and Biggers, J. D. (1993) Culture of preimplantation embryos. Methods Enzymol. 225, 153–164.PubMedCrossRefGoogle Scholar
  23. 23.
    Pedersen, R. A., Wu, K., and Balakier, H. (1986) Origin of the inner cell mass in mouse embryos: cell lineage analysis by microinjection. Dev. Biol. 117, 581–595.PubMedCrossRefGoogle Scholar
  24. 24.
    Inoué, S. and Spring, K. R. (1997) Video Microscopy, Plenum, New York.Google Scholar
  25. 25.
    Ehrig, T., O’Kane, D. J., and Prendergast, F. G. (1995) Green-fluorescent protein mutants with altered fluorescence excitation spectra. FEBS Lett. 365, 163–166.CrossRefGoogle Scholar
  26. 26.
    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. Biotechnology 13, 151–154.PubMedCrossRefGoogle Scholar
  27. 27.
    Heim, R., Prasher, D. C., and Tsien, R. Y. (1994) Wavelength mutations and posttranslational autoxidation of green fluorescent protein. Proc. Natl. Acad. Sci. USA 91, 12,501–12,504.PubMedCrossRefGoogle Scholar
  28. 28.
    Crameri, A., Whitehorn, E. A., Tate, E., and Stemmer, W. P. C. (1996) Improved green fluorescent protein by molecular evolution using DNA shuffling. Nat. Biotechnol. 14, 315–319.PubMedCrossRefGoogle Scholar
  29. 29.
    Anderson, M. T., Tjioe, I. M., Lorincz, M. C., Parks, D. R., Herzenberg, L. A., Nolan, G. P., and Herzenberg, L. A. (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

Copyright information

© Humana Press Inc., Totowa, NJ 2000

Authors and Affiliations

  • Magdalena Zernicka-Goetz
    • 1
  • Jonathon Pines
    • 2
  1. 1.Department of Genetics, Wellcome/CRC InstituteUniversity of CambridgeCambridgeUK
  2. 2.Department of ZoologyWellcome/CRC InstituteCambridgeUK

Personalised recommendations