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Performance of Ad-A-Gene EGFP-Glucocorticoid Receptor (EGFP-GCCR): an adenoviral vector gene delivery system

ur 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).
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