DNA Viruses pp 387-400 | Cite as

Construction of a Gene Inactivation Library for Bovine herpesvirus 1 Using Infectious Clone Technology

  • Timothy J. Mahony
  • Fiona M. McCarthy
  • Jennifer L. Gravel
  • and Peter L. Young
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 292)

Abstract

The application of infectious clone technology to herpesvirus biology has revolutionized the study of these viruses. Previously the ability to manipulate these large DNA viruses was limited to methods dependent on homologous recombination in mammalian cells. However, the construction of herpesvirus infectious clones using bacterial artificial chromosome vectors has permitted the application of powerful bacterial genetics for the manipulation of these viruses. A method is described for the construction and characterization of a gene inactivation library of Bovine herpesvirus 1 using an infectious clone. The method utilizes transposon-mediated gene inactivation, which permits gene inactivation without any prior knowledge of the viral genomic sequence. Furthermore, as the genetic manipulation is performed in bacteria the inactivation of those viral genes that are essential for viral replication is also possible. The method described here can be readily applied to any herpesvirus clone and provides the tools for precise characterization of all the genes contained within a herpesvirus genome.

Key Words

Bovine herpesvirus homologous recombination infectious clone Tn5 transpo-son bacterial artificial chromosome gene deletion gene insertion 

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Copyright information

© Humana Press Inc. 2005

Authors and Affiliations

  • Timothy J. Mahony
    • 1
  • Fiona M. McCarthy
    • 1
  • Jennifer L. Gravel
    • 1
  • and Peter L. Young
  1. 1.Department of Primary IndustriesAgency for Food and Fibre SciencesSt. LuciaAustralia

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