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

Abstract

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.
Pages93-108
Number of pages16
DOIs
StatePublished - 2019

Publication series

NameMethods in Molecular Biology
Volume2023
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Keywords

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

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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  • Cite this

    Rico, A. B., Olson, A. T., & Wiebe, M. S. (2019). Generation of Vaccinia Virus Gene Deletion Mutants Using Complementing Cell Lines. In Methods in Molecular Biology (pp. 93-108). (Methods in Molecular Biology; Vol. 2023). Humana Press Inc.. https://doi.org/10.1007/978-1-4939-9593-6_5