Background: Adenoviral vector is an efficient tool for gene transfer. Protein expression is regulated by a number of
factors, but the regulation by gene copy number remains to be investigated further.
Results: Assessed by flow cytometry, we demonstrated a significant linear correlation between average
fluorescence intensity of green fluorescent protein (GFP) and a wide range of multiplicity of infection (MOI),
spanning from 0.01 to 200. Average GFP intensity was calculated by mean fluorescence intensity (MFI) × percentage
of infection (POI) (MFI × POI) and the correlation was observed in cells transduced with GFP-expressing adenoviral
vector driven either by a cytomegalovirus (CMV) promoter for 3 to 6 h or by a human phosphoglycerate kinase
(PGK) promoter for 18 to 24 h. Factors impacting this linear correlation include MOI of viral vector, strength of
promoter driving GFP expression, cell type transduced and incubation time after gene transfer. We also found
that weak GFP signals could be interfered by background signals, whereas strong GFP signals could overshot the
detection limitation of the flow cytometer and resulted in a deviation from linearity which was prevented by adjusting
the setting in flow cytometer. Moreover, we compared promoter strength as measured by MFI × POI and found that
the relative activity of CMV promoter to PGK promoter was 20 to 47 folds in A549 cells and 32 to > 100 folds in
H1299 cells.
Conclusions: The linear correlation between MFI × POI and a wide range of adenoviral MOI provides an efficient
method to investigate factors regulating protein expression and to estimate virus titers.