Adenoviral Transduction. Comparable values for transduction efficiency were determined by the IN Cell Analyzer 1000 and by flow cytometry . HepG2, HeLa, and U2OS cells transduce efficiently (73%) with SW1353 cells being less susceptible. Data are comparable to those in a previous study, which reported adenoviral-mediated transduction efficiencies for U2OS and SW1353 cells.
Lipid-based Transfection. Comparable values for transfection efficiency were determined by the IN Cell Analyzer 1000 and by flow cytometry (Table 1). EGFP-GCCR plasmid efficiencies using a leading commercial lipid system were lower than equivalent transduction obtained with Ad-A-Gene vectors.
Ad-A-Gene Transduction and Plasmid-based TransfectionComparison in an Assay System. The EGFP-GCCR Ad-A-Gene transduced all cell types tested with consistent efficiencies. In contrast, plasmid- transfection efficiencies exhibited a large variation that was cell type-dependent. HeLa and U2OS cells were more amenable to plasmid-transfection compared to either HepG2 or SW1353 cells (Figure 1).
Data of dexamethasone response of cells transduced with the EGFP-GCCR adenovirus show acceptable S:N ratios and p-values in all cell types. In SW1353 and HepG2 cells (which exhibited lower transduction levels at the MOI used), the Nuclear Trafficking Analysis algorithm was able to resolve an acceptable dexamethasone response.
Plasmid-transfection efficiencies were comparably low in all cell types. Acceptable S:N ratios and p-values were obtained in HeLa and U2OS cells, although significant variation in S:N ratio was observed. In general, poor S:N ratios and p-values were determined for plasmid- transfected HepG2 and SW1353 cells.
Data presented in Figure 2 shows the response of EGFP-GCCR and GRE-NTR to increasing concentrations of dexamethasone. This data demonstrates the successful transduction and development of two Ad-A-Gene vectors into assays for GCCR biology.