Use of a negative selectable marker for rapid selection of recombinant vaccinia virus

Stacy D. White, Kip Conwell, Jeffrey Langland, Bertram Jacobs

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

Vaccinia virus has been a powerful tool in molecular biology and vaccine development. Te relative ease of inserting and expressing foreign genes combined with its broad host range has made it an attractive antigen delivery system against many heterologous diseases. Many diferent approaches have been developed to isolate recombinant vaccinia virus generated from homologous recombination; however, most are time-consuming, ofen requiring a series of passages or specifc cell lines. Herein we introduce a rapid method for isolating recombinants using the antibiotic coumermycin and the in-terferon-associated PKR pathway to select for vaccinia virus recombinants. Tis method uses a negative selection marker in the form of a fusion protein, GyrB-PKR, consisting of the coumermycin dimerization domain of Escherichia coli gyrase subunit B fused to the catalytic domain of human PKR. Coumermycin-dependent dimerization of this protein results in activation of PKR and the phosphorylation of translation initiation factor, eIF2α. Phosphorylation of this factor leads to an inhibition of protein synthesis, and an inhibition of virus replication. In the presence of coumermycin, recombinants are isolated due to the loss of this coumermycin-sensitive gene by homologous recombination. We demonstrate that this method of selection is highly efcient and requires limited rounds of enrichment to isolate recombinant virus.

Original languageEnglish (US)
Pages (from-to)303-309
Number of pages7
JournalBioTechniques
Volume50
Issue number5
DOIs
StatePublished - May 2011

Fingerprint

Vaccinia virus
Viruses
Phosphorylation
Dimerization
Homologous Recombination
Genes
Protein Multimerization
DNA Gyrase
Peptide Initiation Factors
Synthetic Vaccines
Molecular biology
Host Specificity
Virus Replication
Escherichia coli
Molecular Biology
Catalytic Domain
Proteins
Fusion reactions
Chemical activation
Cells

Keywords

  • Coumermycin
  • E3l
  • Gyrase-pkr
  • Recombinant
  • Selection
  • Vaccinia virus

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Biotechnology

Cite this

Use of a negative selectable marker for rapid selection of recombinant vaccinia virus. / White, Stacy D.; Conwell, Kip; Langland, Jeffrey; Jacobs, Bertram.

In: BioTechniques, Vol. 50, No. 5, 05.2011, p. 303-309.

Research output: Contribution to journalArticle

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