Abstract
Genes-encoding marker proteins, which are easily assayable, are useful to monitor cell lineage, gene expression, or promoter activities. In gene-transfer technology such marker genes allow a direct and simple detection of successfully transduced cells. The detection of marker gene products such as β-galactosidase (β-gal), chloramphenicol acetyltransferase (CAT), alkaline phosphatase, or luciferase involves either cell fixation, which kills the cells or antibody-mediated detection, which is time consuming. Drug-resistance genes such as neomycin, puromycin, hygromycin, or zeocin allow a positive selection of transduced cells, but require days to weeks of growth in selective media. Moreover, these genes can change the growth characteristics of the transduced cells through terminal differentiation or can interfere with the expression of the gene of interest (1). Therefore, a marker gene system that provides timely, accurate, and nontoxic detection of successfully transduced living cells would be of great advantage. One interesting candidate gene that fulfills these requirements is the gene-encoding green fluorescent protein (GFP). It was originally isolated from the jellyfish Aquorea victoria. The GFP cDNA consists of 730 bp, which encode a 238 amino acid protein with a molecular weight of 27 kD (2). Wild-type GFP emits a vibrant green fluorescence upon exposure to blue light (450-490 nm). The signal is detectable by fluorescence microscopy and fluorescence-activated cell sorting (FACS) (3). Because the fluorescence of wild-type GFP after excitation is not strong enough for many applications, different variants of GFP have been developed. In one such variant, a point mutation was introduced at amino acid 65 (GFP-S65T) leading to a “red-shifted” excitation maximum with an approximately five-fold stronger fluorescent intensity (4). In a further variant, the “red-shifted” GFP was “humanized” by the introduction of numerous silent mutations that alter the codons to those more commonly used in human genes resulting in the improved translation of the gene (5-7). An additional point mutation at amino acid 64 in which phenylalanine was altered to leucine (F64L) further enhances gene expression (8). GFP has been expressed without cytotoxic effects in different organisms and is of special interest as a marker for monitoring cell lines and gene expression (3). The application of GFP in gene-transfer protocols allows the simple detection of transduced cells and offers the possibility for immediate enrichment of viable transduced cells by FACS (3,9,10). This is of great interest in gene transfer into poorly transducable cells, e.g., hematopoietic stem and progenitor cells.
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References
Valera, A., Perales, J. C., Hatzoglou, M., and Bosch, F. (1994) Expression of the neomycin-resistance (neo) gene induces alterations in gene expression and metabolism. Human Gene Ther. 5, 449–456.
Prasher, D. C., Eckenrode, V. K., Ward, W. W., Prender-Gast, F. G., and Cormier, M. J. (1992) Primary structure of the Aquorea victoria green fluorescent protein. Gene 111, 229–233.
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., Cubitt, A. B., and Tsien, R. Y. (1995) Improved green fluorescence. Nature 373, 663–664.
Levy, J. P., Muldoon, R. R., Zolotukhin, S., and Link, C. J. (1996) Retroviral transfer and expression of a humanized, red shifted green fluorescent protein into human tumor cells. Nature Biotechnol. 14, 610–614.
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.
Muldoon, R. R., Levy, J. P., Kain, S. R., Kitts, P. A., and Link Jr., C. J. (1997) Tracking and quantitation of retroviral-mediated transfer using a completely humanized, red shifted green fluorescent protein gene. BioTechniques 22, 162–165.
Cormack, B. P., Valdivia, R. H., and Falkow, S. (1996) FACS-optimized mutants of the green fluorescent protein (GFP). Gene 173, 33–38.
Kain, S. and Ganguly, S. (1995) Use of fusion genes in mammalian transfection. Overview of genetic reporter systems, in Current Protocols in Molecular Biology (Ausubel, M., Rent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., Struhl, K., eds.), Wiley, New York, unit 9.6.
Dunbar, C. E. and Emmons, R. V. B. (1994) Gene transfer into hematopoietic progenitor and stem cells: progress and problems. Stem Cell 12, 563–576.
Riviere, I. and Sadelain, M. (1997) Methods for the construction of retroviral vectors and the generation of high-titer producers, in Methods in Molecular Medicine: Gene Therapy Protocols (Robbins, P. D., ed.), Humana, Totowa, NJ, pp. 59–78.
Riggs, J. L., Mcallister, R. M., and Lennette, E. H. (1974) Immunofluorescent studies of RD-114 virus replication. J. gen. Virol. 25, 21–29.
Klein, D., Indraccolo, S., von Rombs, K., Amadori, A., Salmons, B., and Günzburg, W. H. (1997) Rapid identification of viable retrovirus-transduced cells using the green fluorescent protein as a marker. Gene Ther. 4, 1256–1260.
Cossett, F. L., Takeuchi, Y., Battini, J. L., Weiss, R. A., and Collins, M. K. (1995) High-titer packaging cells producing recombinant retrovirus resistant to human serum. J. Virol. 69, 7430–7436.
Kaptein, L. C. M., Greijer, A. E., Valerio, D., and van Beusechem, V. W. (1997) Optimized conditions for the production of recombinant amphotropic retroviral vector preparations. Gene Ther. 4, 172–176.
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© 2000 Humana Press Inc., Totowa, NJ
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Uckert, W., Pedersen, L., Günzburg, W. (2000). Green Fluorescent Protein Retroviral Vector. In: Walther, W., Stein, U. (eds) Gene Therapy of Cancer. Methods in Molecular Medicine™, vol 35. Humana Press. https://doi.org/10.1385/1-59259-086-1:275
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DOI: https://doi.org/10.1385/1-59259-086-1:275
Publisher Name: Humana Press
Print ISBN: 978-0-89603-714-4
Online ISBN: 978-1-59259-086-5
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