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Green fluorescent protein: green cells do not always indicate gene expression

Gene Therapy volume 11, pages 882–883 (2004)Cite this article

Green fluorescent protein (GFP) has been used as a reporter molecule for gene expression because it emits green fluorescence after blue-light excitation.1, 2 Inclusion of this gene in a vector can allow rapid selection of successfully transduced cells.3, 4, 5 Production of lentiviral vectors involves cotransfection of vector and helper plasmids into 293T cells. After 48 h, vector particles are harvested from the supernatant and applied to target cells, which are then analysed for gene expression.6 While green fluorescence in the target cells could represent integration of the GFP gene into the target cell genome with subsequent gene expression, passive transfer of GFP from the 293T cell supernatant could also be contributing. This study examined the time course of GFP expression following transduction of hepatocytes in vitro with a GFP-expressing lentiviral vector. Comparisons were made with the time course of GFP expression using a particle that did not contain a GFP gene.

These results suggest that GFP expression in target cells at an early time point does not necessarily signify successful transduction. In the formation of a lentiviral vector particle, it is likely that some protein is passively incorporated into the viral particle and transferred to the target cell. The half-life of unmodified GFP is approximately 26 h;8 thus, it takes several days for the passively transferred protein to degrade leading to an overestimation of transduction achieved at early time points. In gene therapy applications using GFP as an expression marker, it is therefore essential to include appropriate controls and analyse over at least a 7-day period to ensure that the green fluorescence seen reflects true transgene expression.

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  1. Department of Medicine, University of Cambridge, Cambridge, UK

    K L Nash & A M L Lever

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  1. K L Nash
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  2. A M L Lever
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Nash, K., Lever, A. Green fluorescent protein: green cells do not always indicate gene expression. Gene Ther 11, 882–883 (2004). https://doi.org/10.1038/sj.gt.3302246

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  • DOI: https://doi.org/10.1038/sj.gt.3302246

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