A flow cytometric protocol for titering recombinant adenoviral vectors containing the green fluorescent protein
- 378 Downloads
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.
Index EntriesAdenovirus flow cytometry green fluorescent protein recombinant titer
Unable to display preview. Download preview PDF.
- 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
- 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
- 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.Google Scholar