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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online January 7, 2005 as doi:10.1096/fj.04-3180fje. |
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Department of Microbiology and Immunology, Tulane University Medical School, New Orleans, Louisiana, USA
1Correspondence: Department of Veterinary Molecular Biology, 960 Technology Boulevard, Bozeman, MT 59718, USA. E-mail: halford{at}montana.edu
Green fluorescent protein (GFP) has gained widespread use as a tool to visualize spatial and temporal patterns of gene expression in vivo. However, it is not generally accepted that GFP can also be used as a quantitative reporter of gene expression. We report that GFP is a reliable reporter of gene expression in individual eukaryotic cells when fluorescence is measured by flow cytometry. Two pieces of evidence that support this conclusion are that 1) GFP fluorescence increases in direct proportion to the GFP gene copy number delivered to cells by a replication-defective adenovirus vector, Ad.CMV-GFP, and 2) the intensity of GFP fluorescence is directly proportional to GFP mRNA abundance in cells. This is further supported by the fact that the induction of GFP gene expression from two inducible promoters (the TRE and ICP0 promoters) is readily detected by flow cytometric measurement of GFP fluorescent intensity. Collectively, the results of this study indicate that GFP fluorescence is a reliable and quantitative reporter of underlying differences in gene expression.
SPECIFIC AIMS
Since the cloning and enhancement of the green fluorescent protein (GFP), GFP has been widely used as a reporter gene. Despite its power, it is not generally accepted that GFP can be used as a quantitative reporter of promoter activity. Numerous studies suggest that GFP may be useful as a quantitative reporter of gene expression. However, available evidence on this point is diffuse and does not definitively prove whether GFP can be reliably used as a quantitative reporter of gene expression.
The current study was initiated to determine 1) whether differences in GFP fluorescent intensity provide a reliable measure of underlying differences in gene expression when measured by a flow cytometer, and 2) if photographs of GFP-expressing cells taken under a fluorescent microscope can be used to approximate quantitative changes in GFP expression.
PRINCIPAL FINDINGS
GFP fluorescence and GFP mRNA increase in proportion to GFP gene copy number
Vero cells were inoculated with 0 to 1000 pfu per cell of Ad.CMV-GFP, a replication-defective adenovirus that expresses GFP under control of the cytomegalovirus (CMV) major immediate-early promoter. At 24 h postinoculation (p.i.), GFP expression in Vero cells was photographed under a fluorescent microscope; the results suggested that GFP fluorescence increased in intensity as the multiplicity of infection (MOI) of Ad.CMV-GFP was increased (Fig. 1
A). This conclusion was subsequently corroborated by flow cytometry, which indicated that between 0.5 and 46 pfu per cell the average GFP fluorescence increased in direct proportion to the MOI of Ad.CMV-GFP (Fig. 1B
; r2=0.99, as determined by regression analysis). Above an MOI of 100, however, increases in fluorescence deviated from linearity and did not increase in direct proportion to MOI (Fig. 1B
). The average GFP fluorescence was statistically different between each group of cells inoculated with 0.5, 1.0, 2.2, 4.6, 10, 22, 46, 100, 215, 464, or 1000 pfu per cell of Ad.CMV-GFP, determined by a two-way t test. Thus, a single 2.15-fold dilution of Ad.CMV-GFP produced a significant change in GFP fluorescence. Two other independent experiments produced equivalent results.
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To determine whether changes in GFP fluorescence accurately reflect underlying changes in GFP mRNA abundance, Vero cells were inoculated with an MOI of 2.2, 4.6, 10, 22, 46, 100, 215, or 464 pfu per cell of Ad.CMV-GFP. At 24 h p.i., cell monolayers were dissociated with trypsin, and 10% of the cells were analyzed for GFP fluorescence via flow cytometry while total RNA was extracted from the remaining cells for Northern blot analysis. The resulting measurements of GFP fluorescence and GFP mRNA yield were normalized to the maximum signal obtained in each group and plotted relative to one another (Fig. 2
). Regression analysis indicated that the relative level of GFP fluorescence observed in each Ad.CMV-GFP-infected culture was strongly correlated with the relative abundance of GFP mRNA observed in the same culture (Fig. 2)
. Therefore, measurements of GFP fluorescent intensity and GFP mRNA abundance provided equivalent measures of GFP gene expression.
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CONCLUSIONS AND SIGNIFICANCE
In the current study, the GFP gene was evaluated for its capacity to function as a quantitative reporter of gene expression. The GFP gene was placed under the control of three different eukaryotic promoters: 1) a constitutive and highly active CMV immediate-early promoter, 2) a doxycycline-inducible TRE promoter, and a 3) VP16-inducible HSV-1 immediate-early promoter. In the case of Ad.CMV-GFP-infected cells, levels of GFP fluorescence increased in direct proportion to the copy number of GFP genes introduced into Vero cells (Fig. 1)
. In the case of Ad.TRE-GFP-infected Vero cells (coinoculated with Ad.CMV-rtTA), addition of doxycycline to cultures caused an 
20-fold increase in GFP fluorescence relative to Ad.TRE-GFP-infected Vero cells that received no doxycycline (not shown). Finally, in the case of Vero cells transfected with a plasmid that carries an HSV-1 immediate-early promoter GFP reporter gene, increasing MOIs of Ad.TRE-VP16 caused a dose-dependent increase in GFP fluorescence (not shown). Therefore, the outcomes of this study strongly suggest that GFP fluorescence can be used as a reliable and quantitative reporter of underlying changes in gene expression.
In the past decade, the qualitative use of fluorescent reporter proteins has fundamentally changed our understanding of many biological processes, but the quantitative capacity of these proteins has generally gone unnoticed. Based on the results presented here, we conclude that GFP has all of the essential properties of a quantitative reporter protein. Whether GFP genes are delivered to cells via transfection, viral infection, or the generation of stable cell lines or transgenic mice, we envision that flow cytometry can be used as a simple, rapid, and robust means to quantitatively measure GFP reporter gene expression in individual cells derived from these systems. Moreover, given that functional chimeric proteins can be made that bear GFP at the amino or carboxyl terminus, GFP-tagged proteins may prove useful as a way to quantitatively measure the expression of a specific protein in single cells. In summary, we conclude that GFP’s capacity to function as a quantitative reporter provides a powerful new tool to address questions about gene regulation that are best addressed at the single-cell level (e.g., reactivation of HSV-1 in latently infected neurons).
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3180fje;
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