Cytotechnology

, Volume 38, Issue 1–3, pp 87–97 | Cite as

Rapid titration of adenoviral infectivity by flow cytometry in batch culture of infected HEK293 cells

  • Vincent Gueret
  • Juan A. Negrete-Virgen
  • Andrew Lyddiatt
  • Mohamed Al-Rubeai
Article

Abstract

There is a constant and growing interest in exploitingadenoviruses as vectors for gene therapy when transientexpression of a therapeutic protein is necessary. Therequirement for an increased viral titre has prompted asearch for techniques by which this virus may be assayedwith greater speed and simplicity. Conventional plaqueassay for quantification of adenoviral vectors titre incurrent use is laborious and time-consuming (up to 14days). We report herein a method for the monitoring ofadenovirus expressing green fluorescent protein thatincorporates rapid and easy sample handling by means offlow cytometric analysis. Cells (HEK293) were infectedwith adenovirus at various multiplicity of infection(MOI), harvested 17 to 20 h post infection and analysedby flow cytometry. Assumptions were made that onefluorescent cell was infected by a single infectiousparticle at a relatively low MOI. The adenoviral titrewas subsequently estimated from cell analysis in arelatively short time. The results obtained with an E1-complementing cell line (HEK293) were compared with thatobtained using a non-complementing cell line (A549). APoisson distribution successfully modelled the profile ofinfection as a function of MOI. This provided a betterunderstanding of adenoviral infection at the earlieststage possible. Monitoring of GFP fluorescence and viruspropagation in a batch culture of infected cells wassubsequently used as a practical application of thevalidated method.

adenovirus titration adenoviral vectors flowcytometry GFP infectivity 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Blanche F, Cameron B, Barbot A, Ferrero L, Guillemin T, Guyot S, Somarriba S & Bisch D (2000) An improved anion-exchange HPLC method for the detection and purification of adenoviral particles. Gene Ther 7: 1055–1062.Google Scholar
  2. Cooper PD (1961) The plaque assay of animal viruses. Adv Virol Res 8: 319–378.Google Scholar
  3. Côté J, Bourget L, Garnier A & Kamen A (1997) Study of adenovirus production in serum-free 293SF suspension culture by GFP-expression monitoring. Biotechnol Prog 13: 709–714.Google Scholar
  4. Domínguez J, Lorenzo MDM & Blasco R (1998) Green fluorescent protein expressed by a recombinant vaccinia virus permits early detection of infected cells by flow cytometry. J Immunol Methods 220: 115–121.Google Scholar
  5. Eykholt RL, Murray DM & Marvin KW (2000) Accelerated titering of adenoviruses. Biotechniques 28: 870–873.Google Scholar
  6. Galbraith DW, Anderson MT & Herzenberg LA (1999) Flow cytometric analysis and FACS sorting of cells based on GFP accumulation. Methods Cell Biol 58: 315–341.Google Scholar
  7. Kay MA, Liu D & Hoogerbrugge PM (1997) Gene Therapy Proc Natl Acad Sci USA 94: 12744–12746.Google Scholar
  8. Luria SE, Darnell JE Jr, Baltimore D & Campbell A (1978) Titration of viruses. In: General Virology (pp.21–32) John Wiley & Sons, New York.Google Scholar
  9. Maizel JV, White DO & Scharff MD (1968) The polypeptides of adenovirus – I. Evidence for multiple protein components in the virion and a comparison of types 2, 7a and 12. Virology 36: 115–125.Google Scholar
  10. March KL, Madison JE & Trapnell BC (1995) Pharmacokinetics of adenoviral vector-mediated gene delivery to vascular smooth muscle cells: modulation by poloxamer 407 and implications for cardiovascular gene therapy. Hum Gene Ther 6: 41–53.Google Scholar
  11. Mittereder N, March KL & Trapnell BC (1996) Evaluation of the concentration and bioactivity of adenovirus vectors for gene therapy. J Virol 70: 7498–7509.Google Scholar
  12. Murakami P & McCaman MT (1999) Quantitation of adenovirus DNA and virus particles with the PicoGreen fluorescent dye. Anal Biochem 274: 283–288.Google Scholar
  13. Nadeau I, Jacob D, Perrier M & Kamen A (2000) 293SF Metabolic flux analysis during cell growth and infection with an adenoviral vector. Biotechnol Prog 16: 872–884.Google Scholar
  14. Nielsen LK, Smyth GK & Greenfield PF (1992) Accuracy of the end point assay for virus titration. Cytotechnology 8: 231–236.Google Scholar
  15. Nyberg-Hoffman C, Shabram P, Li W, Giroux D & Aguilar-Cordova, E (1997) Sensitivity and reproducibility in adenoviral infectious titer determination. Nat Med 3: 808–811.Google Scholar
  16. Philipson L (1961) Adenovirus assay by the fluorescent cell-counting procedure. Virology 15: 263–268.Google Scholar
  17. Pinteric L & Taylor J (1962) The lowered drop method for the preparation of specimens of partially purified virus lysates for quantitative electron micrographic analysis. Virology 18: 359–371.Google Scholar
  18. Russell WC (2000) Update on adenovirus and its vectors. J Gen Virol 81: 2573–2604.Google Scholar
  19. Shabram PW, Giroux DD, Goudreau AM, Gregory RJ, Horn MT, Huyghe BG, Liu X, Nunnally MH, Sugarman BJ & Sutjipto S (1997) Analytical anion-exchange HPLC of recombinant type-5 adenoviral particles. Hum Gene Ther 8: 453–465.Google Scholar
  20. Subramanian S & Srienc F (1996) Quantitative analysis of transient gene expression in mammalian cells using the green fluorescent protein. J Biotechnol 49: 137–151.Google Scholar
  21. Wang Y & Huang S (2000) Adenovirus technology for gene manipulation and functional studies. DDT 5: 10–16.Google Scholar
  22. Weaver LS & Kadan MJ (2000) Evaluation of adenoviral vectors by flow cytometry. Methods 21: 297–312.Google Scholar
  23. Zhao R, Natarajan A & Srienc F (1999) A flow injection flow cytometry system for on-line monitoring of bioreactors. Biotech Bioeng 62: 609–617.Google Scholar

Copyright information

© Kluwer Academic Publishers 2002

Authors and Affiliations

  • Vincent Gueret
    • 1
  • Juan A. Negrete-Virgen
    • 1
  • Andrew Lyddiatt
    • 1
  • Mohamed Al-Rubeai
    • 1
  1. 1.Department of Chemical EngineeringUniversity of BirminghamBirminghamUK

Personalised recommendations