title: Construction, production, and purification of recombinant adenovirus vectors.
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.
authors.affiliation: Unitat Producció Vectors (UPV), CBATEG, Department of Biochemistry and Molecular Biology, Universitat Autonoma Barcelona, Bellaterra, Spain.
authors.forename: Susana
authors.initials: S
authors.lastname: Miravet
authors.forename: Maria
authors.initials: M
authors.lastname: Ontiveros
authors.forename: Jose
authors.initials: J
authors.lastname: Piedra
authors.forename: Cristina
authors.initials: C
authors.lastname: Penalva
authors.forename: Mercè
authors.initials: M
authors.lastname: Monfar
authors.forename: Miguel
authors.initials: M
authors.lastname: Chillón
mesh_terms: D000260:Adenoviruses, Human / Q000235:genetics* / Q000302:isolation & purification*; D018929:Cell Culture Techniques; D002460:Cell Line; D005822:Genetic Vectors / Q000235:genetics* / Q000302:isolation & purification*; D059765:Homologous Recombination; D006801:Humans; D011995:Recombination, Genetic; D019562:Viral Load; D014771:Virion / Q000302:isolation & purification