Human adenovirus serotype 5 (Ad5) has been widely explored as a gene delivery vector. To achieve highly efficient and specific gene delivery, it is often necessary to re-direct Ad5 tropism. Because the capsid protein fiber plays an essential role in directing Ad5 infection, our laboratory attempted to re-target Ad5 through fiber modification. We have developed two strategies in this regard. One is a bi-specific adaptor protein strategy, in which the adaptor protein is designed to bind both the Ad5 fiber and an alternative cell-surface receptor. Another is genetic modification, in which alternative targeting motifs are genetically incorporated into the fiber knob domain so that the Ad5 vectors can infect cells through the alternative receptors. In this chapter, we will focus on the genetic fiber modification strategy and provide a detailed protocol for generation of fiber-modified Ad5 vectors. A series of techniques/procedures used in our laboratory will be described, which include the generation of fiber-modified Ad5 genome by homologous recombination in a bacterial system, rescuing the modified Ad5 viruses, virus amplification and purification, and virus titration.

Original languageEnglish
Title of host publicationGene Therapy Protocols
Subtitle of host publicationDesign and Characterization of Gene Transfer Vectors
PublisherHumana Press
Number of pages20
ISBN (Print)9781603272476
StatePublished - 2008

Publication series

NameMethods in Molecular Biology
ISSN (Print)1064-3745


  • Adenovirus
  • Adenovirus amplification
  • CsCl gradient purification
  • Fiber modification
  • Gene therapy
  • Homologous recombination
  • Rescuing adenoviral vectors


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