title: Linear correlation between average fluorescence intensity of green fluorescent protein and the multiplicity of infection of recombinant adenovirus.
abstract: 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.
authors.affiliation: Graduate School of Life Science, National Defense Medical Center, Taipei, 11490, Taiwan. x3tsaiyc@yahoo.com.tw.|Institute of Biomodical Sciences, Academia Sinica, No. 128, Sec. 2, Academia Road, Taipei, 11529, Taiwan. x3tsaiyc@yahoo.com.tw.
authors.forename: Yi-Chen
authors.initials: YC
authors.lastname: Tsai
authors.affiliation: Institute of Biomodical Sciences, Academia Sinica, No. 128, Sec. 2, Academia Road, Taipei, 11529, Taiwan. tthu5408@yahoo.com.tw.
authors.forename: Tsung-Huang
authors.initials: TH
authors.lastname: Tsai
authors.affiliation: Institute of Biomodical Sciences, Academia Sinica, No. 128, Sec. 2, Academia Road, Taipei, 11529, Taiwan. 2ping2@yahoo.com.tw.
authors.forename: Chen-Ping
authors.initials: CP
authors.lastname: Chang
authors.affiliation: Institute of Biomodical Sciences, Academia Sinica, No. 128, Sec. 2, Academia Road, Taipei, 11529, Taiwan. sfchen@ibms.sinica.edu.tw.
authors.forename: Shu-Fen
authors.initials: SF
authors.lastname: Chen
authors.affiliation: Graduate School of Life Science, National Defense Medical Center, Taipei, 11490, Taiwan. velociraptorpixy@yahoo.com.tw.|Institute of Biomodical Sciences, Academia Sinica, No. 128, Sec. 2, Academia Road, Taipei, 11529, Taiwan. velociraptorpixy@yahoo.com.tw.
authors.forename: Yen-Ming
authors.initials: YM
authors.lastname: Lee
authors.affiliation: Graduate School of Life Science, National Defense Medical Center, Taipei, 11490, Taiwan. skshyue@ibms.sinica.edu.tw.|Institute of Biomodical Sciences, Academia Sinica, No. 128, Sec. 2, Academia Road, Taipei, 11529, Taiwan. skshyue@ibms.sinica.edu.tw.|School of Chinese Medicine, China Medical University, Taichung, 40402, Taiwan. skshyue@ibms.sinica.edu.tw.
authors.forename: Song-Kun
authors.initials: SK
authors.lastname: Shyue
mesh_terms: D000256:Adenoviridae / Q000502:physiology*; D000257:Adenoviridae Infections / Q000821:virology*; D045744:Cell Line, Tumor; D003587:Cytomegalovirus / Q000502:physiology; D005434:Flow Cytometry; D005453:Fluorescence; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D006801:Humans; D010735:Phosphoglycerate Kinase / Q000378:metabolism; D011401:Promoter Regions, Genetic
chemical_list: D049452:Green Fluorescent Proteins; D010735:Phosphoglycerate Kinase