Linear correlation between average fluorescence intensity of green fluorescent protein and the multiplicity of infection of recombinant adenovirus.

Yi-Chen Tsai,Song-Kun Shyue,Yen-Ming Lee,Tsung-Huang Tsai,Chen-Ping Chang,Shu-Fen Chen

doi:10.1186/s12929-015-0137-z

Abstract

BackgroundAdenoviral 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.ResultsAssessed 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.ConclusionsThe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12929-015-0137-z) contains supplementary material, which is available to authorized users.

Highlights

Adenoviral vector is an efficient tool for gene transfer
Correlation between average green fluorescent protein (GFP) intensity and multiplicity of infection (MOI) of Ad-CMV-GFP in A549 cells To investigate whether the average fluorescence intensity for GFP (MFI × percentage of infection (POI)) delineated in equation (3) was correlated linearly with transfected gene copy number, we used adenoviral vectors carrying the GFP gene driven either by a CMV (Ad-CMV-GFP) or a phosphoglycerate kinase (PGK) (Ad-PGK-GFP) promoter to transduce A549 or H1299 cells for 3 to 48 h and examined the relationship between mean fluorescence intensity (MFI) × POI and MOI 0.01 to 200
In A549 cells transduced with Ad-CMV-GFP, MFI × POI showed a good linear correlation with MOI (R2 = 0.998) for MOI 0.1 to 200 at 3 h post-infection (Figure 2B)

Summary

Introduction

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. Viral vector-derived protein expression is regulated by a number of factors, including the promoter used, gene copy number within the cell type transduced and the availability of cellular machinery for transcription and translation in host cells. For viral vectors carrying the green fluorescent protein (GFP) the percentage of infection (POI) or mean fluorescence intensity (MFI) are considered to be linearly correlated with multiplicity of infection (MOI) under specific conditions [3,4,5,6,7].

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Discussion

Conclusion