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Transient selection during vaccinia virus recombination with insertion vectors without selectable markers

M G Kurilla1

  • 1Department of Pathology, University of Virginia Health Sciences Center, Charlottesville 22908, USA. mgk2r@virginia.edu

Biotechniques
|May 1, 1997
PubMed
Summary

This study introduces a novel transient selection method for vaccinia virus (VV) recombinant production. This technique enhances recombinant virus identification by over sevenfold without needing markers in the insertion vector.

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Area of Science:

  • Virology
  • Molecular Biology
  • Genetic Engineering

Context:

  • Recombinant virus production, particularly vaccinia virus (VV), is often hindered by time-consuming isolation steps.
  • Traditional methods rely on selectable markers integrated into insertion vectors, which are not always practical or desirable.
  • Alternative plaque characterization is laborious and inefficient.

Purpose:

  • To develop a transient selection method for VV recombinant isolation that bypasses the need for selectable markers on the insertion vector.
  • To improve the efficiency of identifying recombinant viruses post-transfection and recombination.

Summary:

  • A novel method involves co-transfecting a guanine phosphoribosyl transferase (gpt) selectable marker on a separate plasmid into VV-infected cells pretreated for gpt selection.

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  • Following recombination, the resulting virus can be plated without further selection, as the transient selection enriches for recombinant viruses.
  • This approach leads to a significant increase, over sevenfold, in the frequency of recombinant viruses within the progeny, simplifying identification.

    • Impact:

    • Streamlines the production of vaccinia virus recombinants, reducing labor and time.
    • Offers a flexible alternative to traditional selectable marker systems for VV engineering.
    • Facilitates more efficient screening and isolation of desired recombinant viruses for research and therapeutic applications.