A flow cytometric protocol for titering recombinant adenoviral vectors containing the green fluorescent protein.
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 absorbance, 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 x 10(9) PFU/mL versus 3.46 +/- 0.76 x 10(9) green fluorescent units/(gfu/mL). The flow cytometric method is complete within 24 h in contrast to the 7 x 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.
Hitt
D C
DC
Department of Surgery, University of Oklahoma Health Sciences Center, Oklahoma City 73190, USA. Daron-Hitt@ouhsc.edu
Booth
J L
JL
Dandapani
V
V
Pennington
L R
LR
Gimble
J M
JM
Metcalf
J
J
D000256:Adenoviridae / Q000737:chemistry / Q000254:growth & development*; D005434:Flow Cytometry / Q000379:methods*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000032:analysis*; D010948:Viral Plaque Assay; D014776:Virus Cultivation
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins