Abstract
Recombinant adenoviruses provide a versatile system for gene expression studies and therapeutic applications. In this chapter, a standard procedure for their generation and small-scale production is described. Homologous recombination in E. coli between shuttle plasmids and full-length adenovirus backbones (E1-deleted) is used for the generation of recombinant adenoviral vectors genomes. The adenovirus genomes are then analyzed to confirm their identity and integrity, and further linearized and transfected to generate a recombinant adenoviral vector in permissive human cells. These vectors are then purified by two sequential CsCl gradient centrifugations and subjected to a chromatography step in order to eliminate the CsCl and exchange buffers. Finally, the viral stock is characterized through the quantification of its viral particle content and its infectivity.
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Miravet, S., Ontiveros, M., Piedra, J., Penalva, C., Monfar, M., Chillón, M. (2014). Construction, Production, and Purification of Recombinant Adenovirus Vectors. In: Chillón, M., Bosch, A. (eds) Adenovirus. Methods in Molecular Biology, vol 1089. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-679-5_12
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DOI: https://doi.org/10.1007/978-1-62703-679-5_12
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Publisher Name: Humana Press, Totowa, NJ
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