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

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
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