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Performance of Ad-A-Gene EGFP-MAPKAP-k2: an adenoviral vector gene delivery system

Key words: adenoviral vector • viral transduction • EGFP • IN Cell Analyzer • gene delivery

Biological assays developed in cultured and primary cells can greatly aid secondary screening as well as lead and target validation, but their development presents several challenges. Establishing stable cell systems to express target genes of interest at detectable levels is time-consuming. In addition, cellular signaling is complex, with data often difficult to interpret reproducibly.

Engineered adenoviral vectors mitigate these challenges by providing facile ways to deliver key genes into target cells. The Ad-A-Gene adenoviral vector gene delivery system rapidly delivers a range of signal pathway “sensors” into mammalian target cells via viral transduction (1, 2). These sensors comprise a panel of cellular genes, each of which is fused as a chimera to either enhanced green fluorescent protein (EGFP) or emerald fluorescent protein (eFP), or a response element controlling the expression of the E. coli B nitroreductase (NTR) reporter gene (3–7). The chimeric genes become transiently expressed within transduced target cells, allowing for the development of cellular assays that exploit the signal readouts from the sensor gene markers.

This application note describes the performance of an EGFP reporter assay for assessing the translocation of mitogen-activated protein kinase-activated protein kinase2 (MAPKAP-k2). MAPK signaling pathways are instrumental in the transduction of extracellular signals into intracellular responses. For instance, one specific set of pathways mediated by p38MAPK, are activated in response to stresses such as heat shock, ultraviolet light, bacterial lipopolysaccharide (LPS) and to pro-inflammatory cytokines, such as tumor necrosis factor alpha (TNFa) and interleukin 6 (IL-6). MAPKAP-k2, a direct substrate of p38MAPK yme active form. J. Med. Chem. 46, 4009–4020 (2003).

8. Rouse, J. et al. A novel kinase cascade triggered by stress and heat shock that stimulates MAPKAP kinase-2 and phosphorylation of the small heat shock proteins. Cell 78, 1027–1037(1994).

9. Stokoe, D. et al. MAPKAP kinase-2; a novel protein kinase activated by mitogen-activated protein kinase. EMBO J. 11, 3985–3994 (1992).

10. Dhawan, P. et al. Critical role of p42/44MAPK activation in anisomycin and hepatocyte growth factor-induced LDL receptor expression: activation of Raf1/MEK-1/p42/44MAPK cascade alone is sufficient to induce LDL receptor expression. Clin. Journal of Lipid Res. 40, 1911–1919 (1999).

11. Kumar, S. et al. Novel homologues of CSBP/p38 MAP kinase: activation, substrate specificity, and sensitivity to Inhibition by pyridinyl imidazoles. BBRC. 235, 533–538 (1997).

12. Sudo, T. et al. p38 mitogen-activated protein kinase plays a key role in regulating MAPKAPK2 expression. BBRC. 337, 415-421 (2005).

back to top , resides in both the nucleus and cytoplasm in non-stimulated cells. Stimulation initiates a series of parallel signaling cascades, leading to direct phosphorylation of MAPKAP-k2 by p38MAPK, and subsequent translocation of phosphorylated MAPKAP-k2 from the cell nucleus to the cytoplasm. Translocation is thought to occur via phosphorylation-mediated molecular unmasking of a nuclear export sequence on MAPKAP-k2 (8–12).

MAPKAP-k2 translocation can be monitored by analyzing target cells transduced with the adenoviral vector, EGFPMAPKAP-k2. By monitoring the cellular position and intensity of the EGFP fluorescent signal from the chimera gene product, the distribution of MAPKAP-k2 within the nucleus and cytoplasm in response to stimuli can be examined. The EGFP-MAPKAP-k2 experiments described here were performed in live- and fixed-cell formats using microplates. Signal monitoring and quantitation of the EGFP-MAPKAP-k2 signal, which was performed on an IN Cell Analyzer 1000, indicated the strength of the cellular response to the chemical stimuli under investigation.

Reagents and instrumentation

Ad-A-Gene EGFP-MAPKAP-k2 GDS20001

VSV-Emerald FP GDS30035

IN Cell Analyzer 1000 25-8010-26

IN Cell Analyzer 1000 Seat License* 25-8098-22

Object Intensity Analysis Module for 25-8010-56
IN Cell Analyzer 1000

Nuclear Trafficking Analysis Module for 25-8010-31
IN Cell Analyzer 1000

IN Cell Analyzer 3000 may also be used for these experiments.

* A seat li cense is a cost-effective single-user or server license that gives access to all ready-to-use Image Analysis Modules provided with IN Cell Analyzer. License holders have access to all appropriate analysis software and more licenses can be purchased as the number of users grows.

Available to seat license holders only.

Additional materials required
FACSCalibur™ system (Becton Dickinson)

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)

Assay buffer 47.7-ml media, 500-µl of 1-M HEPES, 500 µl of 2-mM L-glutamine, 1.3 ml of 7.5% bovine serum albumin (BSA)

1-M HEPES (Sigma)

7.5% BSA (Sigma)

PBS: Dulbecco’s (GIBCO BRL)

Anisomycin, 300 nM, in assay buffer (Sigma)

SB203580-HCl, 100 µM, containing anisomycin (300 nM) in assay buffer (Sigma).

Formalin solution (10%), neutral-buffered, 4% (w/v) formaldehyde (Sigma)

10-mM Hoechst™ 33342 nuclear dye (Molecular Probes)

HeLa, MCF7 (ATCC), and BHK stable (MAPKAP-k2-GFP) (Bioimage) cell lines

Assay method for 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. EGFP-MAPKAP-k2 adenoviral vector 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 48 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 product handling.

* An MOI of 10 infectious units (ifu) per cell in HeLa cells has been calculated as optimum for the EGFP-MAPKAP-k2 assay. Other assay conditions will vary and can be optimized by users.

Assay protocol (fixed-cell format)
On the day of the assay, 300-nm anisomycin was prepared from stocks in assay bufferand added to appropriate “test” wells after medium was removed from the cells. Anisomycin is a protein synthesis inhibitor that activates stress-related MAPKs, namely c-jun NH2-terminal kinase (p46/54JNK), and p38MAPK, in mammalian cells (9). Also included were wells containing both 100-µM SB203580 plus anisomycin. SB203580 is a pyridinyl imidazole MAPK inhibitor, reported to selectively inhibit p38MAPK activity with little or no affect on JNK, ERK, or several other protein kinases (11). Plates were incubated at 37 ºC 5% CO2 for 90 min.

Solutions were decanted from the wells and fixing solution was added (100 µl/well) followed by 15-min incubation at room temperature. Fixing solution was decanted from all wells and the cells were washed with PBS (200 µl/well). Finally, Hoechst nuclear dye (2.5 µM) in PBS was added (100 µl/well) and the wells were incubated for 20 min at room temperature.

Plates were imaged on IN Cell Analyzer 1000 using a 10×objective, 360/40-nm (Hoechst) and 475/20-nm (EGFP) excitation filters, and a 535/20-nm emission filter§. Images were analyzed using the Nuclear Trafficking Analysis Module or Object Intensity Analysis Module. The nuclear/cytoplasmi c (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.

‡ The plates can be stored at 2–8 °C at this stage if imaging is not performed immediately.

§ IN Cell Analyzer 3000 requires 488-nm (EGFP) and 363-nm (Hoechst) excitation laser lines, 535/45-nm and 450/65-nm emission filters.

Assay protocol (kinetic live cell)
Test compound (300-nm anisomycin containing 2.5-µm Hoechst nuclear dye) was added at 100 µl/well to cells transduced as described in the 96-well plate protocol. Appropriate wells were set up containing 100 µl/well of 100-µm SB203580 plus anisomycin and dye. Plates were incubated for 90 min at 37 ºC, 5% CO2.

Cells were imaged on IN Cell Analyzer 1000 using the image acquisition method as described 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.

Optimization of MOI in HeLa cells

To ensure good assay performance, functional cellular assays involving adenoviral transduction require initial determination of the optimal MOI, which is achieved by titrating virus into cells. Figure 1 shows data for transduced HeLa cells treated with drugs at a range of MOI. The data show optimal virus loading of HeLa cells under the described con ditions to be 10 MOI, based on the highest S:N ratio. The magnitude of response (MOR) value represents the relative signal window across the range of MOI employed. Users should choose the MOI that provides the optimum balance between viral load on a cell and acceptable signal.

Viral transduction efficiency measurement from flow cytometry data
Flow cytometry was used to quantitate the EGFP signal from cells transduced with the EGFP-MAPKAP-k2 adenoviral vector. Analysis of the data allowed the transduction efficiency in HeLa cells at three MOI loadings to be calculated (Table 1). HeLa cells were set up at 1 x 106 cells per well of a six-well plate and virus was added at 0 (control), 25, 50, and 100 MOI. After 24-h incubation, 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 in two defined fluorescent signal gating regions (R1 corresponding to low EGFP signal and R2 corresponding to high EGFP signal) with increasing MOI compared to the control. At 25 MOI, > 87% of gated cells fall within R2, compared with < 1.5% in the control.

Drug-induced EGFP-MAPKAP-k2 translocation
Figure 2 shows images of HeLa cells 48 h after transduction with the EGFP-MAPKAP-k2 adenoviral vector at 50 MOI. Cytoplasmic translocation of EGFP-MAPKAP-k2 was observed following treatment with 300-nM anisomycin (Fig 2a). The anisomycin-induced effect was blocked by co-treatment of cells with SB203580, causing EGFP-MAPKAP-k2 to be located predominantly within the nucleus (Fig 2b).

MAPKAP-k2 translocation and drug dose dependence
Figure 3 shows a dose-response curve for anisomycin using data collected 90 min after addition. Measurement of the ratio of signal intensity between the nucleus and cytoplasm shows that EGFP-MAPKAP-k2 translocation occurs in a dose-dependent manner in the presence of anisomycin. An EC50 of 21.12-nM anisomycin was calculated from the data.

Transduction of MCF7 cells with the EGFP-MAPKAP-k2 adenoviral vector
Target cells other than HeLa cells can be transduced by the EGFP-MAPKAP-k2 adenoviral vector. Figure 4 shows data acquired from MCF7 cells transduced with the EGFPMAPKAP-k2 vector. The transduced cells produce fusion proteins that function in a pharmacologically relevant manner. The results with MCF7 cells compare favorably to those obtained from transduced HeLa cells shown in Figure 1. The data for HeLa and MCF7 cells also illustrate key differences between transduction efficiencies in different cell lines. In practice, 10 MOI was considered adequate to achieve an acceptable signal-to-noise ratio for HeLa cells, whereas 50 MOI was found to be more appropriate for MCF7 cells.

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 that the functionality of the transiently expressed gene is comparable, within certain criteria, to equivalent genes expressed in stable cell lines. Figure 5 shows the dose-response to anisomycin of cells from the stable BHK line expressing MAPKAP-k2-GFP (EC50 = 46.66 nM), which compares favorably with earlier data from HeLa cells transiently transfected with the EGFP-MAPKAPk2 adenoviral vector (EC50 = 21.12 nM).

Note: When this experiment was conducted, it was not possible to obtain comparative data from a single cell type. Exercise caution when attempting direct comparisons of data from differing cell types.

Exposure of "null" VSV-Emerald FP adenoviral vector to cells from a stable MAPKAP-k2 cell line
It is important to establish whether exposure of target cells to the adenoviral vector affects the tested biology of the cells. This was achieved by treatment of cells from the stable BHK cell line expressing MAPKAP-k2-GFP with a “null” VSV-Emerald FP adenoviral vector, the latter containing no responding, or active, gene of interest. The data in Figure 6 show that EC50 values for anisomycin were similar (range 63.5–71.6 nM) in both cases, indicating that adenoviral exposure did not affect the measured biology of the stable BHK MAPKAP-k2-GFP expressing cells.

Reproducibility of adenoviral transduction
Transduced HeLa cells transiently expressing EGFPMAPKAP-k2 were treated with varying doses of anisomycin for 90 min prior to fixing, image acquisition, and analysis. The normalized results from three experiments are shown in Figure 7. These data show that adenoviral transduction is both robust and reproducible: there is excellent agreement in EC50 values across the three experiments and goodness-of-fit (R2) is well within statistically acceptable limits.

The EGFP-GCCR chimera, when transiently expressed in host cells using adenoviral vector gene delivery, functions well in a translocation assay upon exposure to pharmacologically relevant stimuli. The response is dose-dependent and produces results that compare favorably to those obtained in a stable cell line equivalent. The system has no effect on the measured biology of target cells and experimental variation is statistically acceptable.

Methods using Ad-A-Gene techno logy to create cell-based assays are more simple than plasmid-based transfection and data can be obtained rapidly. This work also suggests that 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. Graham, F. L. et al. Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J. Gen. Virol. 36, 59–74 (1977).

2. Krougliak, V. and Graham, F. L. Development of cell lines capable of complementing E1, E4, and protein IX defective adenovirus type 5 mutants. Hum. Gene Ther. 6, 1575–1586 (1995).

3. Kozarsky, K. F. and Wilson, J. M. Gene therapy: adenovirus vectors. Curr. Opin. Genet. Dev. 3, 499–503 (1993).

4. Lochmuller, H. et al. Emergence of early region 1-containing replication-competent adenovirus in stocks of replication-defective adenovirus recombinants (delta E1 + delta E3) during multiple passages in 293 cells. Hum. Gene Ther. 5, 1485–1491 (1994).

5. Ng, P. et al. An enhanced system for construction of adenoviral vectors by the two-plasmid rescue method. Hum. Gene Ther. 11, 693–699 (2000).

6. Zhu, J. et al. Characterization of replication-competent adenovirus isolates from large-scale production of a recombinant adenoviral vector. Hum. Gene Ther. 10, 113–121 (1999).

7. Johansson, J., et al. Studies on the nitroreductase prodrug-activating system. Crystal structures of complexes with the inhibitor dicoumarol and dinitrobenzamide prodrugs and of the enz


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