Skip to main content
SpringerLink
Log in

Construction and Isolation of Recombinant Vaccinia Virus Using Genetic Markers

  • Protocol
Vaccinia Virus and Poxvirology

Part of the book series: Methods in Molecular Biology ((MIMB,volume 269))

Abstract

The standard approach for the isolation of vaccinia virus recombinants involves homologous recombination between a transfected plasmid and the replicating viral DNA. In a typical infection/transfection experiment, recombinant viruses only account for a tiny proportion (10−4 to 10−3) of the progeny virus; thus, genetic markers are often included in the transfected plasmid to facilitate the selection of recombinant viruses. This chapter describes in detail two different selection procedures: one relies on plaque formation phenotype using the vaccinia virus gene F13L; the other relies on antibiotic resistance using the Escherichia coli xanthine-guanine phosphoribosyl transferase gene.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 109.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Moss, B. and Flexner, C. (1987) Vaccinia virus expression vectors. Annu. Rev. Immunol. 5, 305–324.

    Article  PubMed  CAS  Google Scholar 

  2. Moss, B. and Flexner, C. (1989) Vaccinia virus expression vectors. Ann. NY Acad. Sci. 569, 86–103.

    Article  PubMed  CAS  Google Scholar 

  3. Moss, B. (1992) Vaccinia virus vectors. Biotechnology 20, 345–362.

    PubMed  CAS  Google Scholar 

  4. Moss, B. (1996) Genetically engineered poxviruses for recombinant gene expression, vaccination, and safety. Proc. Natl. Acad. Sci. USA 93, 11341–11348.

    Article  PubMed  CAS  Google Scholar 

  5. Earl, P. and Moss, B. Expression of proteins in mammalian cells using vaccinia viral vectors, in Current Protocols in Molecular Biology (Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., Struhl, K., eds.), Wiley-Interscience, New York, 1991.

    Google Scholar 

  6. Scheiflinger, F., Dorner, F., and Falkner, F. G. (1992) Construction of chimeric vaccinia viruses by molecular cloning and packaging. Proc. Natl. Acad. Sci. USA 89, 9977–9981.

    Article  PubMed  CAS  Google Scholar 

  7. Merchlinsky, M. and Moss, B. (1992) Introduction of foreign DNA into the vaccinia virus genome by in vitro ligation: recombination-independent selectable cloning vectors. Virology 190, 522–526.

    Article  PubMed  CAS  Google Scholar 

  8. Merchlinsky, M., Eckert, D., Smith, E., and Zauderer, M. (1997) Construction and characterization of vaccinia direct ligation vectors. Virology 238, 444–451.

    Article  PubMed  CAS  Google Scholar 

  9. Chakrabarti, S., Brechling, K., and Moss, B. (1985) Vaccinia virus expression vector: coexpression of beta-galactosidase provides visual screening of recombinant virus plaques. Mol. Cell Biol. 5, 3403–3409.

    PubMed  CAS  Google Scholar 

  10. Panicali, D., Grzelecki, A., and Huang, C. (1986) Vaccinia virus vectors utilizing the beta-galactosidase assay for rapid selection of recombinant viruses and measurement of gene expression. Gene 47, 193–199.

    Article  PubMed  CAS  Google Scholar 

  11. Carroll, M. W. and Moss, B. (1995) E. coli beta-glucuronidase (GUS) as a marker for recombinant vaccinia viruses. Biotechniques 19, 352–354, 356.

    PubMed  CAS  Google Scholar 

  12. Dominguez, J., Lorenzo, M. M., and Blasco, R. (1998) Green fluorescent protein expressed by a recombinant vaccinia virus permits early detection of infected cells by flow cytometry. J. Immunol. Methods 220, 115–121.

    Article  PubMed  CAS  Google Scholar 

  13. Lorenzo, M. M. and Blasco, R. (1998) PCR-based method for the introduction of mutations in genes cloned and expressed in vaccinia virus. Biotechniques 24, 308–313.

    PubMed  CAS  Google Scholar 

  14. Franke, C. A., Rice, C. M., Strauss, J. H., and Hruby, D. E. (1985) Neomycin resistance as a dominant selectable marker for selection and isolation of vaccinia virus recombinants. Mol. Cell Biol. 5, 1918–1924.

    PubMed  CAS  Google Scholar 

  15. Katz, J. B. and Middle, L. A. (1990) Evaluation of thymidine kinase and neomycin phosphotransferase II positive selection systems for recovery of genetically atypical and recombinant DNA vaccine viruses. Biologicals 18, 301–304.

    Article  PubMed  CAS  Google Scholar 

  16. Sanchez-Puig, J. M. and Blasco, R. (2000) Puromycin resistance (pac) gene as a selectable marker in vaccinia virus. Gene 257, 57–65.

    Article  PubMed  CAS  Google Scholar 

  17. Falkner, F. G. and Moss, B. (1988) Escherichia coli gpt gene provides dominant selection for vaccinia virus open reading frame expression vectors. J. Virol. 62, 1849–1854.

    PubMed  CAS  Google Scholar 

  18. Rodriguez, J. F. and Esteban, M. (1989) Plaque size phenotype as a selectable marker to generate vaccinia virus recombinants. J. Virol. 63, 997–1001.

    PubMed  CAS  Google Scholar 

  19. Blasco, R. and Moss, B. (1995) Selection of recombinant vaccinia viruses on the basis of plaque formation. Gene 158, 157–162.

    Article  PubMed  CAS  Google Scholar 

  20. Staib, C., Drexler, I., Ohlmann, M., Wintersperger, S., Erfle, V., and Sutter, G. (2000) Transient host range selection for genetic engineering of modified vaccinia virus Ankara. Biotechniques 28, 1137–1142, 1144–1146, 1148.

    PubMed  CAS  Google Scholar 

  21. Perkus, M. E., Limbach, K., and Paoletti, E. (1989) Cloning and expression of foreign genes in vaccinia virus, using a host range selection system. J. Virol. 63, 3829–3836.

    PubMed  CAS  Google Scholar 

  22. Smith, K. A., Stallard, V., Roos, J. M., Hart, C., Cormier, N., Cohen, L. K., et al. (1993) Host range selection of vaccinia recombinants containing insertions of foreign genes into non-coding sequences. Vaccine 11(1), 43–53.

    Article  PubMed  CAS  Google Scholar 

  23. Blasco, R. and Moss, B. (1991) Extracellular vaccinia virus formation and cell-to-cell virus transmission are prevented by deletion of the gene encoding the 37,000-dalton outer envelope protein. J. Virol. 65, 5910–5920.

    PubMed  CAS  Google Scholar 

  24. Blasco, R. and Moss, B. (1992) Role of cell-associated enveloped vaccinia virus in cell-to-cell spread. J. Virol. 66, 4170–4179.

    PubMed  CAS  Google Scholar 

  25. Boyle, D. B. and Coupar, B. E. H. (1988) A dominant selectable marker for the construction of recombinant poxviruses. Gene 65, 123–128.

    Article  PubMed  CAS  Google Scholar 

  26. Chakrabarti, S., Sisler, J. R., and Moss, B. (1997) Compact, synthetic, vaccinia virus early/late promoter for protein expression. Biotechniques 23, 1094–1097.

    PubMed  CAS  Google Scholar 

  27. Galindo, I., Lorenzo, M. M., and Blasco, R. (2001) Set of vectors for the expression of histidine-tagged proteins in vaccinia virus recombinants. Biotechniques 30, 524–526, 528–529.

    PubMed  CAS  Google Scholar 

  28. Blasco, R., Cole, N. B., and Moss, B. (1991) Sequence analysis, expression, and deletion of a vaccinia virus gene encoding a homolog of profilin, a eukaryotic actin-binding protein. J. Virol. 65, 4598–4608.

    PubMed  CAS  Google Scholar 

  29. Blasco, R. and Moss, B. (1992) Role of cell-associated enveloped vaccinia virus in cell-to-cell virus spread. J. Virol. 66, 4170–4179.

    PubMed  CAS  Google Scholar 

  30. Isaacs, S. N., Kotwal, G. J., and Moss, B. (1990) Reverse guanine phosphoribosyltransferase selection of recombinant vaccinia viruses. Virology 178, 626–630.

    Article  PubMed  CAS  Google Scholar 

  31. Fuerst, T. R., Niles, E. G., Studier, F. W., and Moss, B. (1986) Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proc. Natl. Acad. Sci. USA 83, 8122–8126.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Departamento de Biotecnologíia, Instituto Nacional de Investigacióon y Tecnologíia Agraria y Alimentaria (INIA), Madrid, Spain

    Maríia M. Lorenzo, Inmaculada Galindo & Rafael Blasco

Authors
  1. Maríia M. Lorenzo
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Inmaculada Galindo
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Rafael Blasco
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Division of Infectious Diseases, University of Pennsylvania, Philadelphia, PA

    Stuart N. Isaacs

Rights and permissions

Reprints and permissions

Copyright information

© 2004 Humana Press Inc., Totowa, NJ

About this protocol

Cite this protocol

Lorenzo, M.M., Galindo, I., Blasco, R. (2004). Construction and Isolation of Recombinant Vaccinia Virus Using Genetic Markers. In: Isaacs, S.N. (eds) Vaccinia Virus and Poxvirology. Methods in Molecular Biology, vol 269. Humana Press. https://doi.org/10.1385/1-59259-789-0:015

Download citation

  • DOI: https://doi.org/10.1385/1-59259-789-0:015

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-229-2

  • Online ISBN: 978-1-59259-789-5

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation