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Construction, Production, and Purification of Recombinant Adenovirus Vectors

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Adenovirus

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1089))

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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Author information

Authors and Affiliations

  1. Unitat Producció Vectors (UPV), CBATEG, Department of Biochemistry and Molecular Biology, Universitat Autonoma Barcelona, Bellaterra, Spain

    Susana Miravet, Maria Ontiveros & Mercè Monfar

  2. Department of Biochemistry and Molecular Biology, Unitat Producció Vectors (UPV), CBATEG, Universitat Autonoma Barcelona, Bellaterra, Spain

    Jose Piedra & Cristina Penalva

  3. Institut Català de Recerca i Estudis Avançats (ICREA) and CBATEG-Department of Biochemistry and Molecular Biology, Universitat Autonoma Barcelona, Bellaterra, Barcelona, Spain

    Miguel Chillón

Authors
  1. Susana Miravet
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  2. Maria Ontiveros
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  3. Jose Piedra
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  4. Cristina Penalva
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  5. Mercè Monfar
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  6. Miguel Chillón
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Editor information

Editors and Affiliations

  1. Biochemistry and Molecular Biology, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, Spain

    Miguel Chillón

  2. Biochemistry and Molecular Biology, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, Spain

    Assumpció Bosch

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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

  • Print ISBN: 978-1-62703-678-8

  • Online ISBN: 978-1-62703-679-5

  • eBook Packages: Springer Protocols

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