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Robust functional gene validation by adenoviral vectors: one-step Escherichia coli-Derived Recombinant Adenoviral Genome construction

Gene Therapy volume 11, pages 1599–1605 (2004)Cite this article

Abstract

We describe here a clonal approach for efficient and robust construction of recombinant adenoviral genomes that holds certain advantages over existing approaches. Transgenes of interest are cloned into a small, conditionally replicating plasmid containing the left end of a recombinant adenoviral genome, encompassing pIX coding regions. Transformation of this plasmid into recombination-competent Escherichia coli bearing a plasmid containing the right end of a recombinant adenoviral genome, commencing from pIX coding regions, yields a stable co-integrated plasmid encoding a full adenoviral genome, by virtue of shared homology in pIX coding regions contained in both plasmids. The recombination process yielding the full adenoviral plasmid requires only one step, and always results in the formation of only the desired recombinant adenoviral genome. Thus, no screening is required to identify the correct plasmid encoding the desired recombinant adenoviral genome. In addition, the plasmid encoding the right-hand side of the adenoviral genome is itself incapable of producing contaminating adenovirus. We have successfully employed this approach to generate over 200 recombinant adenoviruses, obtaining only the desired recombinant adenoviral species each time. The process is amenable to medium-to-high-throughput parallel construction of adenoviral genomes, and as such should aid efforts aimed towards high-throughput functional annotation of therapeutic gene targets, which aim to leverage the benefits of adenoviruses as gene delivery and expression vectors.

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Acknowledgements

We gratefully acknowledge the support of the Centre National de la Recherche Scientifique and the European Union Marie Curie Fellowship Programme in carrying out this study. We are also grateful to Patrice Yeh for reviewing the manuscript prior to submission.

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Authors and Affiliations

  1. Aventis Pharma, Functional Genomics, Vitry-sur-Seine Cedex, France

    B Mullan, C Dugué, V Moutard, D Raoux, G Tremp, P Denèfle & J-J Robert

  2. CNRS UMR 8121, Institut Gustave Roussy, Villejuif Cedex, France

    B Mullan, C Dugué, V Moutard, D Raoux, M Perricaudet & J-J Robert

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  1. B Mullan
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  2. C Dugué
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Mullan, B., Dugué, C., Moutard, V. et al. Robust functional gene validation by adenoviral vectors: one-step Escherichia coli-Derived Recombinant Adenoviral Genome construction. Gene Ther 11, 1599–1605 (2004). https://doi.org/10.1038/sj.gt.3302333

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