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Nucleic Acids Research 2005 33(8):e76; doi:10.1093/nar/gni074
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Published online 5 May 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions{at}oupjournals.org

Methods Online

Production of viral vectors using recombinase-mediated cassette exchange

Masakazu Nakano, Kazuhiko Odaka, Yuzuka Takahashi, Masakazu Ishimura, Izumu Saito and Yumi Kanegae*

Laboratory of Molecular Genetics, Institute of Medical Science, University of Tokyo 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan

*To whom correspondence should be addressed. Tel: +81 3 5449 5556; Fax: +81 3 5449 5432; Email: kanegae{at}ims.u-tokyo.ac.jp

Received January 25, 2005. Revised March 23, 2005. Accepted April 7, 2005.

DNA viruses are often used as vectors for foreign gene expression, but large DNA region from cloned or authentic viral genomes must usually be handled to generate viral vectors. Here, we present a unique system for generating adenoviral vectors by directly substituting a gene of interest in a small transfected plasmid with a replaced gene in a replicating viral genome in Cre-expressing 293 cells using the recombinase-mediated cassette exchange (RMCE) reaction. In combination with a positive selection of the viral cis-acting packaging signal connected with the gene of interest, the purpose vector was enriched to 97.5 and 99.8% after three and four cycles of infection, respectively. Our results also showed that the mutant loxP V (previously called loxP 2272), a variant target of Cre used in the RMCE reaction, was useful as a non-compatible mutant to wild-type loxP. This method could be useful for generating not only a large number of adenovirus vectors simultaneously, but also other DNA virus vectors including helper-dependent adenovirus vector.

Present addresses: Masakazu Nakano, Department of Genomic Medical Sciences, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602-8566, Japan.

Masakazu Ishimura, Pharmacology Department, Central Research Laboratories, Kaken Pharmaceutical Co., Ltd, 14, Shinomiya, Minamigawara-cho, Yamashina-ku, Kyoto 607-8042, Japan


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[Abstract] [Full Text] [PDF]


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