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Engineering of a vaccinia virus bacterial artificial chromosome in Escherichia coli by bacteriophage λ–based recombination

Nature Methods volume 2, pages 95–97 (2005)Cite this article

Abstract

The large capacity of vaccinia virus (VAC) for added DNA, cytoplasmic expression and broad host range make it a popular choice for gene delivery, despite the burdensome need for multiple plaque purifications to isolate recombinants. Here we describe how a bacterial artificial chromosome (BAC) containing the entire VAC genome can be engineered in Escherichia coli by homologous recombination using bacteriophage λ–encoded enzymes. The engineered VAC genomes can then be used to produce clonally pure recombinant viruses in mammalian cells without the need for plaque purification.

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Figure 1: Engineering VAC-BAC plasmids by homologous recombination.
Figure 2: Southern blot analysis of the terminal repeat region of VAC-BAC constructs.
Figure 3: Formation and analysis of recombinant viruses expressing RFP and GUS.

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Acknowledgements

We thank D.L. Court for providing mini-λ DNA.

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Authors and Affiliations

  1. Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 4 Center Drive, Bethesda, 20892-0445, Maryland, USA

    Arban Domi & Bernard Moss

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  1. Arban Domi
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  2. Bernard Moss
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Correspondence to Bernard Moss.

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The authors declare no competing financial interests.

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Domi, A., Moss, B. Engineering of a vaccinia virus bacterial artificial chromosome in Escherichia coli by bacteriophage λ–based recombination. Nat Methods 2, 95–97 (2005). https://doi.org/10.1038/nmeth734

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