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di Lullo, G.* ; Soprana, E.* ; Panigada, M.* ; Palini, A.* ; Erfle, V. ; Staib, C. ; Sutter, G. ; Siccardi, A.G.*

Marker gene swapping facilitates recombinant Modified Vaccinia Virus Ankara production by host-range selection.

J. Virol. Methods 156, 37-43 (2009)

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Open Access Green as soon as Postprint is submitted to ZB.

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Modified Vaccinia Virus Ankara (MVA) is employed widely as an experimental and human vaccine vector for its lack of replication in mammalian cells and high expression of heterologous genes. Recombinant MVA technology can be improved greatly by combining transient host-range selection (based on the restoration in MVA of the deleted vaccinia gene K1L) with the differential expression of fluorescent proteins. Recombinant virus results from swapping a red protein gene (in the acceptor virus) with a cassette of the transfer plasmid comprising the transgene and the green marker K1Lgfp (a chimeric gene comprising K1L and EGFP). Recombinant selection is performed in the selective host RK13. Finally, in the non-selective host BHK-21, a single crossover between identical flanking regions excises the marker gene. The three types of viruses involved (red parental, green intermediate and colourless final recombinant) are visualized differentially by fluorescence microscopy or fluoro-imaging of terminal dilution microcultures, leading to a straightforward and efficient purification protocol. This method (Red-to-Green gene swapping) reduces greatly the time needed to obtain marker-free recombinant MVA and increases the reliability of the construction process.

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Publication type Article: Journal article

Document type Scientific Article

Corresponding Author

Keywords Recombinant MVA; Host-range selection; Marker gene swapping; K1Lgfp; Fluorescent proteins; attenuated mva strain; vaccination; vector; immunogenicity; replication; expression; cells; model; dna; propagation

ISSN (print) / ISBN 0166-0934

e-ISSN 0166-0934

Journal Journal of Virological Methods

Quellenangaben Volume: 156, Issue: 1-2, Pages: 37-43

Publisher Elsevier

Non-patent literature Publications

Reviewing status Peer reviewed

Institute(s) Institute of Virology (VIRO)