Key words: adenoviral vector • viral transduction • EGFP • IN Cell Analyzer • gene delivery
There is a requirement in the drug discovery process to investigate lead candidate drugs arising from primary screens and to validate those compounds in suitable biological assays. There is also a need in early-stage drug discovery to identify and measure, as well as modulate the activity of, a plethora of molecular targets involved in signal transduction pathways. Use of biological assays in cultured and primary cells can greatly aid secondary screening and early-stage drug discovery. However, establishing stable cell systems that express target genes of interest at detectable levels can present a genuine challenge. As a way forward, the use of adenoviral vectors to deliver genes and generate transient expression of key gene targets in cells provides an alternative method to techniques such as plasmid transfection.
The Ad-A-Gene adenoviral vector gene delivery system is composed of a panel of genes, each of which is fused to either enhanced green fluorescent protein (EGFP) or Emerald fluorescent protein (eFP), or a response element controlling the expression of the nitroreductase (NTR) reporter gene. Each sensor gene can be rapidly delivered by viral transduction of target cells, resulting in transient gene expression. Signal readouts from the sensor gene markers may then be exploited in the facile development of cellular assays that can report the status of a range of molecular targets at high levels of sensitivity.
This application note presents data from an EGFP translocation assay focusing on the glucocorticoid receptor (GCCR), which is a member of the steroid receptor superfamily. GCCR resides predominantly in the cytoplasm and when glucocorticoids bind to a defined ligand-binding site, the ligand-occupied GCCR translocates to the nucleus.
Translocation of GCCR was monitored by analyzing target cells transduced with the adenoviral vector, EGFP-GCCR. Monitoring the cellular position and signal intensity of the transgene product allowed examination of the distribution of GCCR within the nucleus and cytoplasm. Cellular distribution of EGFP-GCCR protein was shown to alter in response to drugs, such as dexamethasone and prednisolone.
Quantitation of the EGFP signal from the EGFP-GCCR fusion protein within the cells when exposed to chemical stimuli, gives an indication of the potency of those stimuli. Signal monitoring and quantitation was performed on the IN Cell Analyzer 1000 and IN Cell Analyzer 3000 imaging systems.
Reagents and instrumentation
Ad-A-Gene EGFP-Glucocorticoid Receptor GDS 20008
IN Cell Analyzer 1000 25-8010-26
IN Cell Analyzer 1000 Seat License* 25-8098-22
IN Cell Analyzer 3000 25-8010-11
IN Cell Analyzer 3000 Seat License* 63-0055-97
Object Intensity Analysis Module 25-8010-56
(for IN Cell Analyzer 1000)
Object Intensity Analysis Module 63-0048-93
(for IN Cell Analyzer 3000)
Nuclear Trafficking Analysis Module 25-8010-31
(for IN Cell Analyzer 1000)
Nuclear Trafficking Analysis Module 63-0048-94
(for IN Cell Analyzer 3000)
* A seat license is a cost-effective single-user or server license that gives access to all ready to use Image Analysis Modules provided for your IN Cell Analyzer instrument. License holders have access to all appropriate analysis software and more licenses can be purchased as the number of users grows.
Additional materials required
FACSCalibur™ system (Becton Dickson)
DMEM (Sigma), with 10% fetal bovine serum (FBS) (Sigma), 2-mM L-glutamine (Sigma), 100-μg/ml penicillin, and 100-μg/ml streptomycin (Sigma)
Culture medium plus charcoal-stripped FCS (CS-FCS): DMEM (Sigma) with 10% CS-FCS (Sigma)
Dexamethasone (Sigma). 50-mM stock in ethanol; β-estradiol, RU486, progesterone, and prednisolone (Sigma). Stocks in ethanol or DMSO at suitable concentrations.
Dulbecco’s (GIBCO BRL)
Formalin solution (10%), neutral-buffered, 4% (w/v) formaldehyde, (Sigma). Dilute two-fold in PBS.
10-mM Hoechst™ 33342 nuclear dye (Molecular Probes).
HeLa, MCF7, A431, A549 (ATCC), and HEK 293 stable (GFP-GCCR) (GCCR gene obtained by permission from GlaxoSmithKline, and modified in-house into viral gene construct)
Assay method in 96-well microplate format
On the day before the assay, suitable target cells in log-phase growth were detached by treatment with trypsin. Cell concentrations were adjusted to suitable levels with culture medium. The adenoviral vector product was thawed by placing the tube on ice, added to the cell suspension, and mixed to provide the appropriate multiplicity of infection (MOI) for the assay*. The cell and virus suspension was dispensed at 200-μl/well into microplates and incubated for 24 h at 37 °C, 5% CO2.
Note: All adenoviral vector preparations are handled as BSL-2 category reagents. Local safety assessments are made before using the reagent. The product pack literature contains detailed instructions for handling of the product.
* An MOI of 50 to 60 ifu/cell in HeLa cells has been calculated as optimum for the EGFP-GCCR assay. Other assay conditions will vary, and these can be optimized by the end-user.
Assay protocol (fixed cell)
On the day of the assay, test compounds were prepared in assay buffer. Medium was removed from the cells and replaced with medium containing CS-FCS (100-μl/well). Test compounds (e.g., 200-nM dexamethasone) were added* (100-μl/well) and incubated for 30 to 60 min at 37 ºC, 5% CO2.
The solutions were decanted from the wells and fixing solution added (100-μl/well) followed by incubation for 15-min at room temperature. The fixing solution was decanted from all wells, followed by washing with PBS (200-μl/well). Finally, Hoechst nuclear dye (2.5 μM) in PBS was added (100-μl/well), followed by incubation for 20 min at room temperature. The plates were stored at 2–8 °C if imaging was not performed immediately.
Image acquisitions were performed on the IN Cell Analyzer 1000 (using a 10× objective and 360/40-nm (Hoechst) and 475/20-nm (EGFP) excitation filters and 535/20-nm emission filter), or an IN Cell Analyzer 3000 (excitation provided by 488-nm [EGFP] and 363-nm [Hoechst] laser lines and monitored through 535/45-nm and 450/65-nm emission filters).
Images from IN Cell Analyzer 1000 were analyzed using the Nuclear Trafficking Analysis Module or the Object Intensity Analysis Module to quantitatively measure the fluorescence intensity in the cytoplasm and the nucleus of each cell. The nuclear/cytoplasmic (Nuc/Cyt) ratio is the population-averaged ratio of sampled nuclear and cytoplasmic intensities measured in the EGFP signal channel.
* Time-controlled dispensing of compound solutions to the cells can be avoided by completing the assay using live cells and then fixing the cells prior to imaging. Cells should be fixed at the peak translocation time point. Fixing of cells reduces the handling requirements for post-assay material to BSL-1 categorization.
Assay protocol (kinetic live cell)
Test compounds (e.g., 200-nM dexamethasone containing 2.5-μM Hoechst nuclear dye) were added at 100-μl/well to cells in CS-FCS medium (100 μl) that had been transduced 24 h earlier as described previously. Plates were incubated for 30–60 min at 37 ºC, 5% CO2.
Cells were imaged on an appropriate detection instrument, using the image acquisition method as described above for the fixed-cell assay protocol. Analysis of images also followed the method outlined above for the fixed-cell assay protocol.
Note: at all stages of the live-cell assay, the assay plate must be contained within BSL-2 level facilities.
Viral transduction efficiency measurement from flow cytometry data
The primary step to setting up a functional cellular assay involving adenoviral transduction is to determine the optimal MOI by titrating into target cells. We have made use of flow cytometry to quantitate the EGFP signal from cells transduced with the EGFP-GCCR adenoviral vector. Analysis of data allowed calculation of the transduction efficiency in HeLa cells at three MOI loadings (Table 1). HeLa cells were set up at 1 × 106 cells/well of a 6-well plate and virus was added at 0 (control), 25, 50, and 100 MOI. The plates were stored for 24 h and then the cells were trypsinized and resuspended in PBS followed by gated analysis on a FACSCalibur flow cytometer.
The data in Table 1 illustrate the cell-by-cell population distribution into two defined fluorescence signal gating regions (R1 corresponding to low EGFP signal and R2 corresponding to high EGFP signal) with increasing MOI compared to the control. At 100 MOI, > 88% of gated cells fall within R2, compared with < 2% in the control. Acceptable transduction was also achieved at 50 MOI.
Optimization of MOI in HeLa cells
Figure 1 shows MOI optimization data for transduced target HeLa cells treated with 200-nM dexamethasone, with the data processed from images acquired on the IN Cell Analyzer. The signal:noise (S:N) data in Figure 1 show optimal virus loading in HeLa cells under the described conditions to be 62 MOI, based on the highest S:N ratio, although at 16 MOI there was ample response. There is good agreement between the flow cytometry data shown in Table 1, and IN Cell Analyzer data shown in Figure 1. The magnitude of response (MOR) value represents the relative signal window across the range of MOI employed and an MOI offering the optimum balance between viral load on a cell and acceptable signal should be chosen. For this assay, 16–62 MOI was adequate for future experiments using HeLa cells.
Viral transduction efficiency: Time and dose dependence
Time-course data of HeLa cells transduced at 50 MOI with the EGFP-GCCR adenoviral vector and treated at five fixed concentrations of dexamethasone is shown in Figure 2. The data show a maximal response to 200-nM dexamethasone over an optimized 20–30 min drug exposure time.
Dexamethasone-induced EGFP-GCCR translocation
Figure 3 shows images, acquired on the IN Cell Analyzer 3000, of HeLa cells 24 h after transduction with the EGFPGCCR adenoviral vector, and after treatment for 30 min, with buffer medium alone (Fig. 3A) or with 200-nM dexamethasone (Fig. 3B). Figure 3A demonstrates that in the absence of external stimuli, EGFP-GCCR resides in both the nucleus and cytoplasm (at the “snapshot” time of this assay). Figure 3B shows EGFP-GCCR is found predominantly in the nucleus in the presence of 200-nM dexamethasone. These images represent a fraction of the entire field imaged by the IN Cell Analyzer 3000.
GCCR translocation and drug dose dependence
Figure 4 shows a dose response curve for dexamethasone (dex). Data were collected 30 min after the addition of dexamethasone. The data show that translocation of EGFPGCCR, as measured by the ratio of signal intensity between the nucleus and cytoplasm, occurs in the presence of dexamethasone in a dose-dependent manner. An EC50 of 4.14-nM dexamethasone was calculated from the data, which is within acceptable limits for published EC50 values using other cell-based assays (1).
Alternative target cells: Transduction of ten cell types with the EGFP-GCCR adenoviral vector
Earlier data show that HeLa cells can be efficiently transduced with the EGFP-GCCR adenoviral vector. Table 2 summarizes data that show efficiencies of transduction into a broad range of target cell types. The EGFP-GCCR cytoplasmic to nuclear translocation was stimulated by treatment with 200-nM dexamethasone for 30 min. Measurements were performed on the IN Cell Analyzer 1000, IN Cell Analyzer 3000, and by FACS analysis. For the latter, 24 h after transduction, cells were trypsinized and resuspended in PBS followed by analysis on a FACSCalibur flow cytometer.
The data in Table 2 demonstrates the broad tropism of the EGFP-GCCR adenoviral vector in a range of suitable cell types with acceptable transduction efficiencies. In addition, with the selected cell lines, functionality of the transfected gene was demonstrated in many cases even with modest degrees of transduction (e.g. see data for MCF7 cell line transduced for 24 h).
Agonist responses in HeLa cells transduced with the EGFP-GCCR adenoviral vector
Figure 5 shows agonist responses for a range of glucocorticoid compounds and other steroid analogues. The data show differential potency of agonists, with the expected high potency of dexamethasone (mean nucleus to cytoplasm ratio 6.33, SD 1.154) and prednisolone (mean 6.79, SD 0.938) at the GCCR. The high response to RU486 (mean 4.05, SD 0.348), which is reportedly an antagonist, appears to support the observation that RU486, in this system, is functioning as an activator of translocation, as has been noted before (2). Use of the EGFP-GCCR fusion protein enables facile identification of this phenomenon.
Function of gene sensors: Adenoviral transduction vs stable cell lines
The use of adenoviral vectors to generate the transient expression of key genes can avoid the laborious and time-consuming establishment of stable cell lines. It is therefore important to demonstrate that the functionality of the transiently expressed gene is comparable, within certain criteria, to equivalent genes expressed in stable cell lines.
Figure 6 summarizes the response to dexamethasone of target HeLa cells transiently transfected, at a range of MOI, with the EGFP-GCCR adenoviral vector. This was compared to equivalent responses in stable HEK 293 cell lines expressing GFP-GCCR. The data show MOI-dependent functional responses in target HeLa cells that have been transiently transfected with the EGFP-GCCR adenoviral vector. Maximum response, as measured by the nuclear to cytoplasm intensity ratio, was at 40 MOI. However, at all MOI values tested in this experiment, EC50 values calculated for the corresponding assays were comparable, indicating similar sensitivities to the detection of agonists across the range of MOI values employed.
Dose response (EC50 values) to dexamethasone in transduced cells compared favourably to that in stable cell lines, and similar Hill slope values were generated that indicate single-binding site stoichiometry in both cases.
Note: Setup of this experiment required use of different cell types and this should be considered when attempting to make direct comparisons of the resulting data.
Reproducibility of adenoviral transduction
Figure 7 shows normalized results obtained from three experiments in which transduced HeLa cells transiently expressing EGFP-GCCR were treated with varying doses of dexamethasone. The data show that adenoviral transduction is both robust and reproducible and that there is excellent agreement in EC50 values across the three experiments. Goodness-of-fit (R2) is well within acceptable limits.
The EGFP-GCCR chimera, when transiently expressed in host cells after adenoviral transduction, functions well in a translocation assay upon exposure to pharmacologically relevant stimuli. The response is both time and dose dependent. Use of the EGFP-GCCR adenoviral vector shows broad tropism of transduction in a range of target cells with subsequent assay results comparing favorably to those obtained in a stable cell line expressing GFP-tagged GCCR. Experimental variation, when carrying out adenoviral transduction followed by functional evaluation, is statistically acceptable.
The sequential process of viral transduction, exposure of cells to reagents, and data acquisition can be transcribed into suitable automated and integrated workflow systems with standard liquid-handling dispensing units as well as image capture and data analysis workstations.
1. Zhang, S. and Daniellsen, M. Steroid structures and activities [Online] http://nrr.georgetown.edu/NRR/nrrhome.html (1995).
2. Pariante, C. M., et al., The steroid receptor antagonists RU40555 and RU486 activate glucocorticoid receptor translocation and are not excreted by the steroid hormones transporter in L929 cells. J. Endocrinol. 169 (2), 309320 (2001).
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