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  • Viral Transfer Technology
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High throughput production, screening and analysis of adeno-associated viral vectors

Gene Therapy volume 7, pages 924–929 (2000)Cite this article

Abstract

Recombinant adeno-associated viruses (rAAV) are promising candidates as gene vectors, as they transduce non-dividing cells and permit lasting transgene expression in a wide spectrum of tissues. In this paper, we describe a robust procedure for the high throughput production, screening and characterization of rAAV vectors. The technology includes the production of rAAV from rapid small scale plasmid preparations and the analysis of virus productivity (physical and infectious particles) and activity (transgene expression, replication). rAAV are produced by triple transfection (rAAV plasmid and AAV- and adenovirus (Ad)-helper plasmids) on 293 human embryo kidney (HEK) cells. The titers of physical and infectious particles are obtained by dot blot hybridization and by a serial dilution assay, followed by either dot blot hybridization or real-time PCR, respectively. rAAV can be produced and characterized from plasmid mixtures containing as little as 1/100 productive molecules. Experiments on rAAV replication kinetics and Ad helper functions are discussed. All steps are performed in 96-well microtiter plates. The process is reproducible, high throughput, linear and ready for automation.

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Acknowledgements

We thank Karin Jooss, Eric Kremer, Philippe Moullier and Nestor Soria for critically reading of the manuscript; Nasser Armande and Pascal Martinez for the computer program developed for the tangential hyperbole-based analysis; and for kindly providing mini-prep rAAV-lacZ plasmid (Pascale Bouille) Ad2, Ad5, ts-Ad adenoviruses, rep+/cap+ HeLa cells and pAAV-lacZ (Philippe Moullier and Anna Salvetti), CAV-1 and CAV-2 (Eric Kremer) and pAB11, pACG.2 and pXX6 plasmids (R Jude Samulsky).

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  1. Généthon III, 1 rue de l'Internationale, Evry, 91002, France

    L Drittanti, C Rivet, P Manceau, O Danos & M Vega

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  1. L Drittanti
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Drittanti, L., Rivet, C., Manceau, P. et al. High throughput production, screening and analysis of adeno-associated viral vectors. Gene Ther 7, 924–929 (2000). https://doi.org/10.1038/sj.gt.3301191

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