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Green fluorescent protein as a reporter in translational assays
ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 336 (2005) 135–137 www.elsevier.com/locate/yabio
Notes & Tips
Green Xuorescent protein as a reporter in translational assays Xue-Qing Wang, Thomas Hadwen, Joseph A. Rothnagel¤ Department of Biochemistry and Molecular Biology, The Institute for Molecular Bioscience and The Centre for Functional and Applied Genomics, University of Queensland, Brisbane, Qld. 4072, Australia Received 19 April 2004
The bacterial chloramphenicol acetyltransferase (CAT)1 and WreXy luciferase enzymes have been widely used as reporters of expression activity in promoter and translation assays. Although these assays are generally highly sensitive, safe, rapid, and easy to perform, limitations on the use of these reporters still remain. Typically the measurement of reporter activity involves cell lysis and/or chemical or immunoreactions, which can introduce variability between samples and assays. In addition, these assays normally employ internal control plasmids to account for transfection eYciency, and the resulting data usually require normalization to either total protein levels or cell number. Moreover, dilution is frequently required for samples with high levels of product so as not to exceed the limit for reliable quantiWcation, and calibration curves are generally required for each assay or series of assays. We have recently used green Xuorescent protein (GFP) to measure the eVect of complex 5⬘UTRs on the eYciency of translation at a main AUG using Xow cytometry [1,2]. In this paper, we detail this assay and compare it with the traditional reporters CAT and luciferase and with human growth hormone (hGH) in both transiently and stably transfected cells. GFP, originally isolated from the jellyWsh Aequorea victoria, has been used extensively in cell-based protein localization studies and more recently as a transcriptional reporter for gene expression assays [3–6]. However, the advantages of using a GFP-Xow-cytometry-based assay have not been reported in detail. To *
Corresponding author. Fax: +61 7 3365 4699. E-mail address: [email protected] (J.A. Rothnagel). 1 Abbreviations used: GFP, green Xuorescent protein; 5⬘-UTR, 5⬘ untranslated region; hGH, human growth hormone; CAT, chloramphenicol acetyltransferase; ORF, open reading frame; ELISA, enzymelinked immunosorbent assay. 0003-2697/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2004.09.007
compare GFP with commonly used reporters, we constructed mammalian expression vectors containing either the -UTR or the -UTR of GLI1 as the 5⬘-UTR [1] linked to GFP, CAT, luciferase, or hGH. The CAT, luciferase, and hGH open reading frames (ORFs) were ligated into AgeI and NotI restriction sites of the pEGFP-N1 expression vector (Clontech) replacing GFP in constructs with the -UTR or -UTR used in our previous study [1] (Fig. 1A). The integrity of all constructs was conWrmed by sequence analysis. The constructs were transfected into the cell lines HaCaT [7], HeLa [8], and CHO [9] using LipofectAMINE 2000 reagent (Invitrogen). The reporter assays were conducted either on cell lysates or conditioned media 48 h posttransfection using commercially available detection kits: CAT ELISA (Roche), Steady-Glo Luciferase Assay System (Promega), and Growth Hormone ELISA (Bioclone). Due to product saturation it was necessary to dilute the -UTR samples to ensure that the results could be read oV the linear portion of the standard calibration curve. Relative luciferase units were measured for 5 s in a Wallac Trilux 1450 microbeta luminometer and both CAT and hGH ELISAs were measured on a microtiter plate reader (Spectramax 250) at recommended wavelengths. The results from at least three independent transfections in three diVerent cell lines for these reporters showed that the -UTR inhibited translation of the reporters as expected, although there were considerable diVerences in the relative values produced in each of these assay systems (Fig. 1B). To identify a possible source of the variation in translation eYciency observed with these reporters in transient transfections, we used the Flp-In (Invitrogen) system to generate stable cell lines containing the GLI1 5⬘-UTRs linked to GFP or hGH. This system integrates a single copy of the gene of interest into the genome at a
136
Notes & Tips / Anal. Biochem. 336 (2005) 135–137
Fig. 1. Comparison of CAT, luciferase, hGH, and GFP reporters in translation assays. (A) Schematic showing constructs containing either -UTR or -UTR with CAT, luciferase, hGH, or GFP. The -UTR consists of three noncoding exons (1, 1a, and 1b) and contains three independent upstream ORFs (see [1] for details). The -UTR consists of one noncoding exon and contains no upstream ATGs/ORFs. (B) Analysis of the relative translational eYciency of - and -UTRs by CAT, luciferase, hGH, and GFP in transiently transfected cells. All values represent the ratios of -UTR to UTR of CAT, luciferase, and hGH levels in CHO, HaCaT, and HeLa cell lines. Both CAT and hGH were transfected and assayed in duplicates. Luciferase and GFP were assayed four times for each experiment. Each experiment was repeated independently for a minimum of three times. Error bars represent the variation of the mean.
speciWc location, thereby avoiding any confounding aVects on gene expression because of copy number or site of integration. Since the only diVerence between these constructs is the sequence of the 5⬘-UTR, any diVerences in reporter levels can be attributed to the speciWc 5⬘-UTR used. Flp-In expression vectors pcDNA5/ FRT (Invitrogen) were constructed with the -UTR or -UTR linked to GFP or hGH. The Flp-In expression vectors were cotransfected with the pOG44 vector (a Flp recombinase expression plasmid) into the Flp-In-CHO host cell line (Invitrogen) using LipofectAMINE 2000 (Invitrogen). Three days posttransfection the cells were subcultured at a density of less than 25% conXuency and selected in medium containing hygromycin B. The hygromycin-resistant foci from each well were then pooled and the entire population of cells from each well was analyzed using a FACSCalibur (Becton–Dickinson) single-laser instrument with an emission wavelength of 488 nm. Three parameters were simultaneously acquired; forward scatter and side scatter were obtained as indicators of individual cell size and cell surface integrity and used to exclude cell debris from healthy populations. Quantitative analysis of GFP levels in viable cells was then carried out using one parameter (FL1), which detects Xuorescence between 515 and 545 nm. To ensure that the results were reproducible, four independent wells containing stably integrated GFP constructs were used and assayed three times over a 3month period following stable selection. There was very little heterogeneity in GFP expression levels in these samples as shown by the shape of the histograms of GFP Xuorescence intensities (Fig. 2A), indicating that most cells produced a similar amount of the reporter. Moreover, these data conWrmed the earlier transient transfection results observed for the - and -UTR constructs
Fig. 2. Analysis of the relative translation eYciency of -UTR and UTR using GFP and hGH as reporters in stably transfected CHO cells. (A) Fluorescence intensity histograms compiled from a negative control sample, and the - and -UTR GFP constructs. (B) Quantitative analysis of relative translational eYciency of -UTR to -UTR. The error bars represent the variation between assays.
[1]. We also obtained comparable results using hGH as the reporter in the Flp-In expression cell line, supporting the data obtained for GFP in both transient and stable
Notes & Tips / Anal. Biochem. 336 (2005) 135–137
transfection. For both systems, a 20-fold increase in reporter production was observed for the -UTR construct compared with the -UTR (Fig. 2B). By comparison with the GFP-based assay, the assays for CAT, luciferase, and hGH involve signiWcantly more handling, which can contribute to the variation observed within and between experiments. Moreover, the linear portion of the standard calibration curve for these assays is relatively small and is easily exceeded in samples with high levels of the reporter. In the present study the samples expressing elevated reporter levels were diluted by severalfold in all three assays. In addition, these assays cannot discriminate between transfected and nontransfected cells and they require normalization of expression values by either cell number or protein concentration. In contrast, the GFP-based Xow cytometry assay is much simpler, quicker, and more reliable than the other reporter systems. Since GFP Xuorescence does not require cofactors, substrates, or additional gene products, GFP production can be measured directly without the need for cell lysis and exogenous enzymatic or chemical reactions. Therefore, errors that could result from these procedures are eliminated. Furthermore, this assay acquires the Xuorescence for each cell in a given sample, resulting in statistical calculations with higher signiWcance for the whole population. Moreover, the dynamic range of GFP Xuorescence determined by Xow cytometry is essentially unlimited. We routinely set background Xuorescence at approximately 1 and allow an increase for positive Xuorescence of up to 104-fold. Not only does Xow cytometry accommodate high levels of GFP activity but it can also reliably detect very low levels of Xuorescence [2] and easily discriminates between positive Xuorescent cells and negative (background Xuorescence) cells, making the GFPbased assay a useful alternative to luciferase where increased sensitivity is required. Such a dynamic range is
137
unique to the GFP-based assay and is not possible with the other reporter systems. In summary, the GFP-based Xow cytometry assay provides the expression proWle of each positive cell in a given sample and directly provides transfection eYciency and the mean of expression in a positive-cell population. The sensitivity and dynamic range of the GFP assay makes it particularly well suited to translation studies. References [1] X.-Q. Wang, J.A. Rothnagel, Post-transcriptional regulation of the GLI1 oncogene by the expression of alternative 5⬘ untranslated regions, J. Biol. Chem. 276 (2001) 1311–1316. [2] X.-Q. Wang, J.A. Rothnagel, 5⬘-untranslated regions with multiple upstream AUG codons can support low-level translation via leaky scanning and reinitiation, Nucleic Acids Res. 32 (2004) 1382–1391. [3] M. ChalWe, Y. Tu, G. Euskirchen, W.W. Ward, D.C. Prasher, Green Xuorescent protein as a marker for gene expression, Science 263 (1994) 802–805. [4] A.B. Cubitt, R. Heim, S.R. Adams, A.E. Boyd, L.A. Gross, R.Y. Tsien, Understanding, improving and using green Xuorescent proteins, Trends Biochem. Sci. 20 (1995) 448–455. [5] B. Ludin, A. Matus, GFP illuminates the cytoskeleton, Trends Cell Biol. 8 (1998) 72–77. [6] A.-L. Ducrest, M. Amacker, J. Lingner, M. Nabholz, Detection of promoter activity by Xow cytometric analysis of GFP reporter expression, Nucleic Acids Res. 30 (2002) e65. [7] P. Boukamp, R.T. Petrussevska, D. Breitkreutz, J. Hornung, A. Markham, N.E. Fusenig, Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line, J. Cell Biol. 106 (1988) 761–771. [8] G.O. Gey, W.D. CoVman, M.T. Kubicek, Tissue culture studies of the proliferative capacity of cervical carcinoma and normal epithelium, Cancer Res. 12 (1952) 264–265. [9] T.T. Puck, S.J. Cieciura, A. Robinson, Genetics of somatic mammalian cells III. Long-term cultivation of euploid cells from human and animal subjects, J. Exp. Med. 108 (1958) 945–956.
Notes & Tips
Green Xuorescent protein as a reporter in translational assays Xue-Qing Wang, Thomas Hadwen, Joseph A. Rothnagel¤ Department of Biochemistry and Molecular Biology, The Institute for Molecular Bioscience and The Centre for Functional and Applied Genomics, University of Queensland, Brisbane, Qld. 4072, Australia Received 19 April 2004
The bacterial chloramphenicol acetyltransferase (CAT)1 and WreXy luciferase enzymes have been widely used as reporters of expression activity in promoter and translation assays. Although these assays are generally highly sensitive, safe, rapid, and easy to perform, limitations on the use of these reporters still remain. Typically the measurement of reporter activity involves cell lysis and/or chemical or immunoreactions, which can introduce variability between samples and assays. In addition, these assays normally employ internal control plasmids to account for transfection eYciency, and the resulting data usually require normalization to either total protein levels or cell number. Moreover, dilution is frequently required for samples with high levels of product so as not to exceed the limit for reliable quantiWcation, and calibration curves are generally required for each assay or series of assays. We have recently used green Xuorescent protein (GFP) to measure the eVect of complex 5⬘UTRs on the eYciency of translation at a main AUG using Xow cytometry [1,2]. In this paper, we detail this assay and compare it with the traditional reporters CAT and luciferase and with human growth hormone (hGH) in both transiently and stably transfected cells. GFP, originally isolated from the jellyWsh Aequorea victoria, has been used extensively in cell-based protein localization studies and more recently as a transcriptional reporter for gene expression assays [3–6]. However, the advantages of using a GFP-Xow-cytometry-based assay have not been reported in detail. To *
Corresponding author. Fax: +61 7 3365 4699. E-mail address: [email protected] (J.A. Rothnagel). 1 Abbreviations used: GFP, green Xuorescent protein; 5⬘-UTR, 5⬘ untranslated region; hGH, human growth hormone; CAT, chloramphenicol acetyltransferase; ORF, open reading frame; ELISA, enzymelinked immunosorbent assay. 0003-2697/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2004.09.007
compare GFP with commonly used reporters, we constructed mammalian expression vectors containing either the -UTR or the -UTR of GLI1 as the 5⬘-UTR [1] linked to GFP, CAT, luciferase, or hGH. The CAT, luciferase, and hGH open reading frames (ORFs) were ligated into AgeI and NotI restriction sites of the pEGFP-N1 expression vector (Clontech) replacing GFP in constructs with the -UTR or -UTR used in our previous study [1] (Fig. 1A). The integrity of all constructs was conWrmed by sequence analysis. The constructs were transfected into the cell lines HaCaT [7], HeLa [8], and CHO [9] using LipofectAMINE 2000 reagent (Invitrogen). The reporter assays were conducted either on cell lysates or conditioned media 48 h posttransfection using commercially available detection kits: CAT ELISA (Roche), Steady-Glo Luciferase Assay System (Promega), and Growth Hormone ELISA (Bioclone). Due to product saturation it was necessary to dilute the -UTR samples to ensure that the results could be read oV the linear portion of the standard calibration curve. Relative luciferase units were measured for 5 s in a Wallac Trilux 1450 microbeta luminometer and both CAT and hGH ELISAs were measured on a microtiter plate reader (Spectramax 250) at recommended wavelengths. The results from at least three independent transfections in three diVerent cell lines for these reporters showed that the -UTR inhibited translation of the reporters as expected, although there were considerable diVerences in the relative values produced in each of these assay systems (Fig. 1B). To identify a possible source of the variation in translation eYciency observed with these reporters in transient transfections, we used the Flp-In (Invitrogen) system to generate stable cell lines containing the GLI1 5⬘-UTRs linked to GFP or hGH. This system integrates a single copy of the gene of interest into the genome at a
136
Notes & Tips / Anal. Biochem. 336 (2005) 135–137
Fig. 1. Comparison of CAT, luciferase, hGH, and GFP reporters in translation assays. (A) Schematic showing constructs containing either -UTR or -UTR with CAT, luciferase, hGH, or GFP. The -UTR consists of three noncoding exons (1, 1a, and 1b) and contains three independent upstream ORFs (see [1] for details). The -UTR consists of one noncoding exon and contains no upstream ATGs/ORFs. (B) Analysis of the relative translational eYciency of - and -UTRs by CAT, luciferase, hGH, and GFP in transiently transfected cells. All values represent the ratios of -UTR to UTR of CAT, luciferase, and hGH levels in CHO, HaCaT, and HeLa cell lines. Both CAT and hGH were transfected and assayed in duplicates. Luciferase and GFP were assayed four times for each experiment. Each experiment was repeated independently for a minimum of three times. Error bars represent the variation of the mean.
speciWc location, thereby avoiding any confounding aVects on gene expression because of copy number or site of integration. Since the only diVerence between these constructs is the sequence of the 5⬘-UTR, any diVerences in reporter levels can be attributed to the speciWc 5⬘-UTR used. Flp-In expression vectors pcDNA5/ FRT (Invitrogen) were constructed with the -UTR or -UTR linked to GFP or hGH. The Flp-In expression vectors were cotransfected with the pOG44 vector (a Flp recombinase expression plasmid) into the Flp-In-CHO host cell line (Invitrogen) using LipofectAMINE 2000 (Invitrogen). Three days posttransfection the cells were subcultured at a density of less than 25% conXuency and selected in medium containing hygromycin B. The hygromycin-resistant foci from each well were then pooled and the entire population of cells from each well was analyzed using a FACSCalibur (Becton–Dickinson) single-laser instrument with an emission wavelength of 488 nm. Three parameters were simultaneously acquired; forward scatter and side scatter were obtained as indicators of individual cell size and cell surface integrity and used to exclude cell debris from healthy populations. Quantitative analysis of GFP levels in viable cells was then carried out using one parameter (FL1), which detects Xuorescence between 515 and 545 nm. To ensure that the results were reproducible, four independent wells containing stably integrated GFP constructs were used and assayed three times over a 3month period following stable selection. There was very little heterogeneity in GFP expression levels in these samples as shown by the shape of the histograms of GFP Xuorescence intensities (Fig. 2A), indicating that most cells produced a similar amount of the reporter. Moreover, these data conWrmed the earlier transient transfection results observed for the - and -UTR constructs
Fig. 2. Analysis of the relative translation eYciency of -UTR and UTR using GFP and hGH as reporters in stably transfected CHO cells. (A) Fluorescence intensity histograms compiled from a negative control sample, and the - and -UTR GFP constructs. (B) Quantitative analysis of relative translational eYciency of -UTR to -UTR. The error bars represent the variation between assays.
[1]. We also obtained comparable results using hGH as the reporter in the Flp-In expression cell line, supporting the data obtained for GFP in both transient and stable
Notes & Tips / Anal. Biochem. 336 (2005) 135–137
transfection. For both systems, a 20-fold increase in reporter production was observed for the -UTR construct compared with the -UTR (Fig. 2B). By comparison with the GFP-based assay, the assays for CAT, luciferase, and hGH involve signiWcantly more handling, which can contribute to the variation observed within and between experiments. Moreover, the linear portion of the standard calibration curve for these assays is relatively small and is easily exceeded in samples with high levels of the reporter. In the present study the samples expressing elevated reporter levels were diluted by severalfold in all three assays. In addition, these assays cannot discriminate between transfected and nontransfected cells and they require normalization of expression values by either cell number or protein concentration. In contrast, the GFP-based Xow cytometry assay is much simpler, quicker, and more reliable than the other reporter systems. Since GFP Xuorescence does not require cofactors, substrates, or additional gene products, GFP production can be measured directly without the need for cell lysis and exogenous enzymatic or chemical reactions. Therefore, errors that could result from these procedures are eliminated. Furthermore, this assay acquires the Xuorescence for each cell in a given sample, resulting in statistical calculations with higher signiWcance for the whole population. Moreover, the dynamic range of GFP Xuorescence determined by Xow cytometry is essentially unlimited. We routinely set background Xuorescence at approximately 1 and allow an increase for positive Xuorescence of up to 104-fold. Not only does Xow cytometry accommodate high levels of GFP activity but it can also reliably detect very low levels of Xuorescence [2] and easily discriminates between positive Xuorescent cells and negative (background Xuorescence) cells, making the GFPbased assay a useful alternative to luciferase where increased sensitivity is required. Such a dynamic range is
137
unique to the GFP-based assay and is not possible with the other reporter systems. In summary, the GFP-based Xow cytometry assay provides the expression proWle of each positive cell in a given sample and directly provides transfection eYciency and the mean of expression in a positive-cell population. The sensitivity and dynamic range of the GFP assay makes it particularly well suited to translation studies. References [1] X.-Q. Wang, J.A. Rothnagel, Post-transcriptional regulation of the GLI1 oncogene by the expression of alternative 5⬘ untranslated regions, J. Biol. Chem. 276 (2001) 1311–1316. [2] X.-Q. Wang, J.A. Rothnagel, 5⬘-untranslated regions with multiple upstream AUG codons can support low-level translation via leaky scanning and reinitiation, Nucleic Acids Res. 32 (2004) 1382–1391. [3] M. ChalWe, Y. Tu, G. Euskirchen, W.W. Ward, D.C. Prasher, Green Xuorescent protein as a marker for gene expression, Science 263 (1994) 802–805. [4] A.B. Cubitt, R. Heim, S.R. Adams, A.E. Boyd, L.A. Gross, R.Y. Tsien, Understanding, improving and using green Xuorescent proteins, Trends Biochem. Sci. 20 (1995) 448–455. [5] B. Ludin, A. Matus, GFP illuminates the cytoskeleton, Trends Cell Biol. 8 (1998) 72–77. [6] A.-L. Ducrest, M. Amacker, J. Lingner, M. Nabholz, Detection of promoter activity by Xow cytometric analysis of GFP reporter expression, Nucleic Acids Res. 30 (2002) e65. [7] P. Boukamp, R.T. Petrussevska, D. Breitkreutz, J. Hornung, A. Markham, N.E. Fusenig, Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line, J. Cell Biol. 106 (1988) 761–771. [8] G.O. Gey, W.D. CoVman, M.T. Kubicek, Tissue culture studies of the proliferative capacity of cervical carcinoma and normal epithelium, Cancer Res. 12 (1952) 264–265. [9] T.T. Puck, S.J. Cieciura, A. Robinson, Genetics of somatic mammalian cells III. Long-term cultivation of euploid cells from human and animal subjects, J. Exp. Med. 108 (1958) 945–956.