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

Journal of Biomedical Science

Song-Kun Shyue²  Yen-Ming Lee¹  Shu-Fen Chen¹  Chen-Ping Chang¹  Tsung-Huang Tsai¹  Yi-Chen Tsai¹ 

[1] Institute of Biomodical Sciences, Academia Sinica, No. 128, Sec. 2, Academia Road, Taipei 11529, Taiwan;School of Chinese Medicine, China Medical University, Taichung 40402, Taiwan

关键词: Promoter activity;    Linear correlation;    Average fluorescence intensity;    Flow cytometry;    Adenovirus;   

Others  :  1213952
DOI  :  10.1186/s12929-015-0137-z

 received in 2014-12-24, accepted in 2015-04-21,  发布年份 2015

【 摘 要 】

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.

【 授权许可】

   
2015 Tsai et al.; licensee BioMed Central.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Figure 7.

Figure 8.

【 参考文献 】

• [1]Crystal RG. Adenovirus: the first effective in vivo gene delivery vector. Hum Gene Ther. 2014; 25(1):3-11.
• [2]Majhen D, Calderon H, Chandra N, Fajardo CA, Rajan A, Alemany R et al.. Adenovirus-based vaccines for fighting infectious diseases and cancer: progress in the field. Hum Gene Ther. 2014; 25(4):301-17.
• [3]Musco ML, Cui S, Small D, Nodelman M, Sugarman B, Grace M. Comparison of flow cytometry and laser scanning cytometry for the intracellular evaluation of adenoviral infectivity and p53 protein expression in gene therapy. Cytometry. 1998; 33(3):290-6.
• [4]Park MT, Lee GM. Rapid titer assay of adenovirus containing green fluorescent protein gene using flow cytometric analysis. Bioprocess Eng. 2000; 22:403-6.
• [5]Weaver LS, Kadan MJ. Evaluation of adenoviral vectors by flow cytometry. Methods. 2000; 21(3):297-312.
• [6]Gueret V, Negrete-Virgen JA, Lyddiatt A, Al-Rubeai M. Rapid titration of adenoviral infectivity by flow cytometry in batch culture of infected HEK293 cells. Cytotechnology. 2002; 38(1-3):87-97.
• [7]Li Z, Ling L, Liu X, Laus R, Delcayre A. A flow cytometry-based immuno-titration assay for rapid and accurate titer determination of modified vaccinia Ankara virus vectors. J Virol Methods. 2010; 169(1):87-94.
• [8]Guo Z, Yang NS, Jiao S, Sun J, Cheng L, Wolff JA et al.. Efficient and sustained transgene expression in mature rat oligodendrocytes in primary culture. J Neurosci Res. 1996; 43(1):32-41.
• [9]Chang TC, Huang CJ, Tam K, Chen SF, Tan KT, Tsai MS et al.. Stabilization of hypoxia-inducible factor-1{alpha} by prostacyclin under prolonged hypoxia via reducing reactive oxygen species level in endothelial cells. J Biol Chem. 2005; 280(44):36567-74.
• [10]Chen SF, Wu CH, Lee YM, Tam K, Tsai YC, Liou JY et al.. Caveolin-1 interacts with Derlin-1 and promotes ubiquitination and degradation of cyclooxygenase-2 via collaboration with p97 complex. J Biol Chem. 2013; 288(46):33462-9.
• [11]Bergelson JM, Cunningham JA, Droguett G, Kurt-Jones EA, Krithivas A, Hong JS et al.. Isolation of a common receptor for Coxsackie B viruses and adenoviruses 2 and 5. Science. 1997; 275(5304):1320-3.
• [12]Hung SC, Lu CY, Shyue SK, Liu HC, Ho LL. Lineage differentiation-associated loss of adenoviral susceptibility and Coxsackie-adenovirus receptor expression in human mesenchymal stem cells. Stem Cells. 2004; 22(7):1321-9.
• [13]Lonsdale R, Pau MG, Oerlemans M, Ophorst C, Vooys A, Havenga M et al.. A rapid method for immunotitration of influenza viruses using flow cytometry. J Virol Methods. 2003; 110(1):67-71.
• [14]Drayman N, Kler S, Ben-nun-Shaul O, Oppenheim A. Rapid method for SV40 titration. J Virol Methods. 2010; 164(1-2):145-7.
• [15]Drayman N, Oppenheim A. Rapid titration of viruses by flow cytometry. Curr Protoc Cell Biol. 2011; 51:26.11.1-7.