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

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 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.

journal_name

Mol Biotechnol

journal_title

Molecular biotechnology

authors

Hitt DC,Booth JL,Dandapani V,Pennington LR,Gimble JM,Metcalf J

doi

10.1385/MB:14:3:197

keywords:

subject

Has Abstract

pub_date

2000-03-01 00:00:00

pages

197-203

issue

3

eissn

1073-6085

issn

1559-0305

pii

MB:14:3:197

journal_volume

14

pub_type

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