Abstract
The unique spectral properties and versatility of autofluorescent proteins have facilitated their widespread use in flow cytometric applications. The ability to analyze heterologous fluorescent protein expression conveniently and noninvasively by individually interrogating cells has facilitated increasingly more sophisticated experimental designs to address important biological questions. Improved multilaser flow cytometers have allowed the fluorescent protein field to flourish by permitting high-speed, multiparametric analysis of biological samples. Fluorescent proteins are well suited for either transient or stable expression analysis. Therefore, achieving efficient gene transfer and expression in cells by transfection or viral transduction is paramount to the optimal use of fluorescent proteins in flow cytometry. The archetypal autofluorescent protein, enhanced green fluorescent protein (eGFP), can be used successfully in combination with other fluorescent protein variants. Two such variants, Cerianthus sp. orange fluorescent protein (cOFP) and a fast maturing variant of Discosoma sp. red protein (DsREDExpress), are well suited for flow cytometric applications in combination with eGFP and do not require special filters for optimal excitation and detection.
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References
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.
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.
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.
Cormack, B. P., Valdivia, R. H., and Falkow, S. (1996) FACS-optimized mutants of the green fluorescent protein (GFP). Gene 173, 33–38.
Yang, T. T., Cheng, L., and Kain, S. R. (1996) Optimized codon usage and chromophore mutations provide enhanced sensitivity with the green fluorescent protein. Nucleic Acids Res. 24, 4592–4593.
Shaner, N. C., Steinbach, P. A., and Tsien, R. Y. (2005) A guide to choosing fluorescent proteins. Nat. Methods 2, 905–909.
Hawley, T. S., Telford, W. G., Ramezani, A., and Hawley, R. G. (2001) Fourcolor flow cytometric detection of retrovirally expressed red, yellow, green, and cyan fluorescent proteins. Biotechniques 30, 1028–1034.
Bevis, B. J. and Glick, B. S. (2002) Rapidly maturing variants of the Discosoma red fluorescent protein (DsRed). Nat. Biotechnol. 20, 83–87.
Ip, D., Chan, S.-H., Allen, M., Bycroft, M., Wan, D., and Wong, K.-B. (2004) Crystallization and preliminary crystallographic analysis of a novel orange fluorescent protein from the Cnidaria tube anemone Cerianthus sp. Acta Crystallograph. D60, 340–341.
Jordan, M., Schallhorn, A., and Wurm, F. M. (1996) Transfecting mammalian cells: optimization of critical parameters affecting calcium-phosphate precipitate formation. Nucleic Acids Res. 24, 596–601.
Pear, W. (1996) Transient transfection methods for high-titre retroviral supernatants, in Current Protocols in Molecular Biology (Ausubel, F. M., Brent, R., Kingston, R. E., et al., eds.), John Wiley & Sons, New York, pp. 9.11.11–9.11.18.
Miller, A. D. and Rosman, G. J. (1989) Improved retroviral vectors for gene transfer and expression. Biotechniques 7, 980–982, 984–986, 989–990.
Bartz, S. R. and Vodicka, M. A. (1997) Production of high-titer human immunodeficiency virus type 1 pseudotyped with vesicular stomatitis virus glycoprotein. Methods 12, 337–342.
Koldej, R., Cmielewski, P., Stocker, A., Parsons, D. W., and Anson, D. S. (2005) Optimisation of a multipartite human immunodeficiency virus based vector system; control of virus infectivity and large-scale production. J. Gene Med. 7, 1390–1399.
Forestell, S. P., Dando, J. S., Bohnlein, E., and Rigg, R. J. (1996) Improved detection of replication-competent retrovirus. J. Virol. Methods 60, 171–178.
Luthman, H. and Magnusson, G. (1983) High efficiency polyoma DNA transfection of chloroquine treated cells. Nucleic Acids Res. 11, 1295–1308.
Hanenberg, H., Xiao, X. L., Dilloo, D., Hashino, K., Kato, I., and Williams, D. A. (1996) Colocalization of retrovirus and target cells on specific fibronectin fragments increases genetic transduction of mammalian cells. Nat. Methods 2, 876–882.
Rasko, J. E. J. (1999) Reporters of gene expression: autofluorescent proteins, in Current Protocols in Cytometry (Robinson, J. P., Zbigniew, P. N., Dressler, L. G., et al., eds.), John Wiley & Sons, Inc, New York, pp. 9.12.11–19.12.16.
Alexander, I. E., Russell, D. W., and Miller, A. D. (1997) Transfer of contaminants in adeno-associated virus vector stocks can mimic transduction and lead to artifactual results. Hum. Gene Ther. 8, 1911–1920.
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© 2007 Humana Press Inc., Totowa, NJ
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Bailey, C.G., Rasko, J.E.J. (2007). Autofluorescent Proteins for Flow Cytometry. In: Anson, D.S. (eds) Reporter Genes. Methods in Molecular Biology, vol 411. Humana Press. https://doi.org/10.1007/978-1-59745-549-7_7
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DOI: https://doi.org/10.1007/978-1-59745-549-7_7
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