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Herpes Simplex Virus pp 131–151Cite as

oHSV Genome Editing by Means of galK Recombineering

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 2060))

Abstract

Since the cloning of the herpes simplex virus (HSV) genome as BAC (bacterial artificial chromosome), the genetic engineering of the viral genome has become readily feasible. The advantage is that the modification of the animal virus genome is carried out in bacteria, with no replication or production of viral progeny, and is separated from the reconstitution or regeneration of the recombinant virus in mammalian cells. This allows an easy engineering of essential genes, as well. Many technologies have been developed for herpesvirus BAC engineering. In our hands the most powerful is galK recombineering that exploits a single marker (galK) for positive and negative selection and PCR amplicons for seamless modification in the desired genome locus. Here we describe the engineering of the HSV recombinant BAC 115 by the insertion of a heterologous cassette for the expression of murine interleukin 12 (mIL12) in the intergenic sequence between US1 and US2 ORFs.

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Acknowledgments

This work was supported by European Research Council (ERC) Advanced Grant number 340060, VII framework program to G.C.-F., by RFO (University of Bologna) to L.M. and T.G, and by Fondi Pallotti to T.G.

Competing interests: G.C.-F. owns shares in Nouscom Srl. B.P. is currently an employee of Nouscom Srl. G.C.-F. and L.M. receive equity payments from Amgen. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Authors and Affiliations

  1. Department of Pharmacy and Biotechnology, University of Bologna, Bologna, Italy

    Laura Menotti

  2. Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Bologna, Italy

    Valerio Leoni, Valentina Gatta, Andrea Vannini, Simona Pepe, Tatiana Gianni & Gabriella Campadelli-Fiume

  3. Nouscom Srl, Rome, Italy

    Biljana Petrovic

Authors
  1. Laura Menotti
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  2. Valerio Leoni
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  3. Valentina Gatta
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  4. Biljana Petrovic
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  5. Andrea Vannini
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  6. Simona Pepe
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  7. Tatiana Gianni
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  8. Gabriella Campadelli-Fiume
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Corresponding author

Correspondence to Gabriella Campadelli-Fiume .

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Editors and Affiliations

  1. Department of Biomedical Sciences, Macquarie University, Sydney, NSW, Australia

    Russell J. Diefenbach

  2. Institute of Virology, University of Zurich, Zürich, Switzerland

    Cornel Fraefel

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Menotti, L. et al. (2020). oHSV Genome Editing by Means of galK Recombineering. In: Diefenbach, R., Fraefel, C. (eds) Herpes Simplex Virus . Methods in Molecular Biology, vol 2060. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9814-2_7

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  • DOI: https://doi.org/10.1007/978-1-4939-9814-2_7

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9813-5

  • Online ISBN: 978-1-4939-9814-2

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