Generation of Vaccinia Virus Gene Deletion Mutants Using Complementing Cell Lines

Amber B. Rico, Annabel T. Olson, Matthew S. Wiebe

Research output: Chapter in Book/Report/Conference proceedingChapter


This protocol describes how to couple two techniques, the generation of complementing cells lines and production of viral deletion mutants, to rapidly construct novel tools for poxvirus analysis. Specifically, the production and utilization of a complementing cell line expressing a poxvirus gene of interest are critical for the generation of poxvirus mutants in which essential genes are disrupted. Complementing cells are also valuable for the characterization of vaccinia genes in the absence of infection. Here, we detail the process of isolating vaccinia virus deletion mutants. Deletion mutant generation involves homologous recombination between replicating viral DNA and transfected DNA followed by selection and screening on a complementing cell line that provides the deleted gene in trans. Finally, deletion is confirmed by polymerase chain reaction, sequencing, and functional assays if available.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Number of pages16
StatePublished - 2019

Publication series

NameMethods in Molecular Biology
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029


  • Complementing cells
  • Deletion mutant
  • Essential gene
  • Fluorescence
  • Vaccinia

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics


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