A selectable and excisable marker system for the rapid creation of recombinant poxviruses

Julia L. Rintoul, Jiahu Wang, Don B. Gammon, Nicholas J. van Buuren, Kenneth Garson, Karen Jardine, Michele Barry, David H. Evans, John C. Bell

Research output: Contribution to journalArticle

21 Citations (Scopus)

Abstract

Background: Genetic manipulation of poxvirus genomes through attenuation, or insertion of therapeutic genes has led to a number of vector candidates for the treatment of a variety of human diseases. The development of recombinant poxviruses often involves the genomic insertion of a selectable marker for purification and selection purposes. The use of marker genes however inevitably results in a vector that contains unwanted genetic information of no therapeutic value. Methodology/Principal Findings: Here we describe an improved strategy that allows for the creation of marker-free recombinant poxviruses of any species. The Selectable and Excisable Marker (SEM) system incorporates a unique fusion marker gene for the efficient selection of poxvirus recombinants and the Cre/loxP system to facilitate the subsequent removal of the marker. We have defined and characterized this new methodological tool by insertion of a foreign gene into vaccinia virus, with the subsequent removal of the selectable marker. We then analyzed the importance of loxP orientation during Cre recombination, and show that the SEM system can be used to introduce site-specific deletions or inversions into the viral genome. Finally, we demonstrate that the SEM strategy is amenable to other poxviruses, as demonstrated here with the creation of an ectromelia virus recombinant lacking the EVM002 gene. Conclusion/Significance: The system described here thus provides a faster, simpler and more efficient means to create clinic-ready recombinant poxviruses for therapeutic gene therapy applications.

Original languageEnglish (US)
Article numbere24643
JournalPLoS One
Volume6
Issue number9
DOIs
StatePublished - Sep 8 2011

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Poxviridae
Genes
therapeutics
Viruses
Ectromelia virus
Vaccinia virus
genetic markers
gene therapy
genome
genes
Insertional Mutagenesis
Viral Genome
Gene Fusion
Gene therapy
Therapeutics
genetic engineering
human diseases
Genetic Therapy
Genetic Recombination
Purification

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

Cite this

Rintoul, J. L., Wang, J., Gammon, D. B., van Buuren, N. J., Garson, K., Jardine, K., ... Bell, J. C. (2011). A selectable and excisable marker system for the rapid creation of recombinant poxviruses. PLoS One, 6(9), [e24643]. https://doi.org/10.1371/journal.pone.0024643

A selectable and excisable marker system for the rapid creation of recombinant poxviruses. / Rintoul, Julia L.; Wang, Jiahu; Gammon, Don B.; van Buuren, Nicholas J.; Garson, Kenneth; Jardine, Karen; Barry, Michele; Evans, David H.; Bell, John C.

In: PLoS One, Vol. 6, No. 9, e24643, 08.09.2011.

Research output: Contribution to journalArticle

Rintoul, JL, Wang, J, Gammon, DB, van Buuren, NJ, Garson, K, Jardine, K, Barry, M, Evans, DH & Bell, JC 2011, 'A selectable and excisable marker system for the rapid creation of recombinant poxviruses', PLoS One, vol. 6, no. 9, e24643. https://doi.org/10.1371/journal.pone.0024643
Rintoul, Julia L. ; Wang, Jiahu ; Gammon, Don B. ; van Buuren, Nicholas J. ; Garson, Kenneth ; Jardine, Karen ; Barry, Michele ; Evans, David H. ; Bell, John C. / A selectable and excisable marker system for the rapid creation of recombinant poxviruses. In: PLoS One. 2011 ; Vol. 6, No. 9.
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