Construction, production, and purification of recombinant adenovirus vectors

S. Miravet, M. Ontiveros, J. Piedra, C. Penalva, M. Monfar, M. Chillon

Research output: Contribution to journalArticleResearchpeer-review

6 Citations (Scopus)

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. © 2014 Springer Science+Business Media, LLC.
Original languageEnglish
Pages (from-to)159-173
JournalMethods in Molecular Biology
Volume1089
DOIs
Publication statusPublished - 1 Jan 2014

Keywords

  • Adenoviral vector
  • Adenoviral vector production
  • Adenovirus construction
  • Adenovirus purification
  • Homologous recombination

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