Skip to main content
SpringerLink
Log in

A flow cytometric protocol for titering recombinant adenoviral vectors containing the green fluorescent protein

  • Research
  • Published:
Molecular Biotechnology Aims and scope Submit manuscript

Abstract

As the use of adenoviral vectors in gene therapy protocols increases, there is a corresponding need for rapid, accurate, and reproducible titer methods. Multiple methods currently exist for determining titers of recombinant adenoviral vector, including optical absorbence, electron microscopy, fluorescent focus assay, and the “gold standard” plaque assay. This paper introduces a novel flow cytometric method for direct titer determination that relies on the expression of the green fluorescent protein (GFP), a tracking marker incorporated into several adenoviral vectors. This approach was compared to the plaque assay using 10−4-to 10−6-fold dilutions of a cesium-chloride-purified, GFP expressing adenovirus (AdEasy+GFP+GAL). The two approaches yielded similar titers: 3.25±1.85×109 PFU/mL versus 3.46±0.76×109 green fluorescent units/(gfu/mL). The flow cytometric method is complete within 24 h in contrast to the 7×10 days required by the plaque assay. These results indicate that the GFU/mL is an alternative functional titer method for fluorescent-tagged adenoviral vectors.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Berkner, K. (1988) Development of adenovirus vectors for the expression of heterologous genes. Biotechniques. 6, 616–624.

    PubMed  CAS  Google Scholar 

  2. Dulver, K. W. (1996) Gene Therapy: A Primer for Physicians, Mary Ann Liebert, Larchmont, NY.

    Google Scholar 

  3. Mitterder, N., March, K.L., and Trapnell, B.C. (1996) Evaluation of the concentration and bioactivity of adenovirus vectors for gene therapy. Journal of Virology 70, 7498–7509.

    Google Scholar 

  4. Philipson, L. (1961) Adenovirus assay by the fluorescent cell-counting procedure. Virology 15, 263–268.

    Article  PubMed  CAS  Google Scholar 

  5. Lawrence, W.C., and Ginsberg, H.S. (1967) Intracellular uncoating of type 5 adenovirus deoxyribonucleic acid. Journal of Virology 1, 851–867.

    PubMed  CAS  Google Scholar 

  6. Strayer, D.S., Duan, L.-X., Ozaki, I., Milano, J., Bobraski, L.E., and Bagasra, O. (1997) Titering replication-defective virus for use in gene transfer. Biotechniques 2, 447–450.

    Google Scholar 

  7. Zhu, J., Grace, M., Casale, J., Chang, A.T.-I., Musco, M.L., Bordens, R., et al. (1999) Characterization of replication-competent adenovirus isolates from largescale production of a recombinant adenoviral vector. Human Gene Therapy 10, 113–121.

    Article  PubMed  CAS  Google Scholar 

  8. Nyberg-Hoffman, C., Shabram, P., Li, W., Giroux, D., and Aguilar-Cordova, E. (1997) Sensitivity and reproducibility in adenoviral intectious titer determination. Nature Medicine 3, 808–811.

    Article  PubMed  CAS  Google Scholar 

  9. Maizel, J.V., White, D., and Scharff, M.D. (1968) The polypeptide of adenovirus. I. Evidence for multiple protein components in the virion and a comparison of types 2, 7a, and 12. Virology 36, 115–125.

    Article  PubMed  CAS  Google Scholar 

  10. Musco, M.L., Cui, S., Small, D., Nodelman, M., Sugarman, B., and Grace, M. (1998) Comparison of flow cytometry and laser scanning cytometry for the intracellular evaluation of adenoviral infectivity and p53 protein expression in gene therapy. Cytometry 33, 290–296.

    Article  PubMed  CAS  Google Scholar 

  11. Chalfie, M., Tu, Y., Euskirchen, G., Ward, W.W., and Prasher, D.C. (1994) Green fluorescent protein as a marker for gene expression. Science 263, 802–805.

    Article  PubMed  CAS  Google Scholar 

  12. Inouye, S., and Tsuji, F.I. (1994) Aequora green fluorescent protein. Expression of the gene and fluorescent characteristics of the recombinant protein. FEBS Letters 341, 277–280.

    Article  PubMed  CAS  Google Scholar 

  13. He, T.-C., Zhou, S., da Costa, L.T., Yu, J., Kinzler, K.W., and Vogelstein, B. (1998) A simplified system for generating recombinant adenoviruses. Proceedings of the National Academy of Science USA 95, 2509–2514.

    Article  CAS  Google Scholar 

  14. Cheng, L., Fu, J., Tsukamoto, A., and Hawley, R.G. Use of green fluorescent protein variants to monitor gene transfer and expression in mammalian cells. Nature Biotechnology 14, 606–609.

  15. Subramanian, S., and Srienc, F. (1996) Quantitative analysis of transient gene expression in mammalian cells using the green fluorescent protein. Journal of Biotechnology 49, 137–151.

    Article  PubMed  CAS  Google Scholar 

  16. Levy, J.P., Muldoon, R.R., Zolotukhin, S., and Link, C.J. Jr. (1996) Retroviral transfer and expression of a humanized, red-shifted green fluorescent protein into human tumor cells. Nature Biotechnology 14, 610–614.

    Article  PubMed  CAS  Google Scholar 

  17. Dominguez, J., del Mar Lorenzo, M., and Blasco, R. (1998) Green fluorescent protein expressed by a recombinant vaccinia virus permits early detection of infected cells by flow cytometry. Journal of Immunological Methods 220, 115–121.

    Article  PubMed  CAS  Google Scholar 

  18. Ropp, J.D., Donahue, C.J., Wolfgang-Kimball, D., Hooley, J.J., Chin, J.Y.W., Hoffman, R.A., Cuthbertson, R.A., et al. (1995) Aequora green fluorescent protein analysis by flow cytometry. Cytometry 21, 309–317.

    Article  PubMed  CAS  Google Scholar 

  19. Ropp, J.D., Donahue, C.J., Wolfgang-Kimball, D., Hooley, J.J., Chin, J.Y.W., Cuthbertson, R.A., et al. (1996) Aequora green fluorescent protein: simultaneous analysis of wild-type and blue-fluorescing mutant by flow cytometry. Cytometry 24, 284–288.

    Article  PubMed  CAS  Google Scholar 

  20. Lybarger, L., Dempsey, D., Franek, K.J., and Chervenak, R. (1996) Rapid generation and flow cytometric analysis of stable GFP-expressing cells. Cytometry 25, 211–220.

    Article  PubMed  CAS  Google Scholar 

  21. Graham, F.L., Smiley, J., Russell, W.C., and Nairn, R. (1977) Characteristics of a human cell line transformed by DNA from human adenovirus Type 5. Journal of General Virology 36, 59–74.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Surgery, University of Oklahoma Health Sciences Center, P.O. Box 26901, 73190, Oklahoma City, OK

    Daron C. Hitt

Authors
  1. Daron C. Hitt
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. J. Leland Booth
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Viji Dandapani
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Larry R. Pennington
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jeffrey M. Gimble
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jordan Metcalf
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Daron C. Hitt.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Hitt, D.C., Booth, J.L., Dandapani, V. et al. A flow cytometric protocol for titering recombinant adenoviral vectors containing the green fluorescent protein. Mol Biotechnol 14, 197–203 (2000). https://doi.org/10.1385/MB:14:3:197

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1385/MB:14:3:197

Index Entries

Search

Navigation