title: Production of viral vectors using recombinase-mediated cassette exchange.
abstract: 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.
authors.affiliation: Laboratory of Molecular Genetics, Institute of Medical Science, University of Tokyo 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.
authors.forename: Masakazu
authors.initials: M
authors.lastname: Nakano
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Odaka
authors.forename: Yuzuka
authors.initials: Y
authors.lastname: Takahashi
authors.forename: Masakazu
authors.initials: M
authors.lastname: Ishimura
authors.forename: Izumu
authors.initials: I
authors.lastname: Saito
authors.forename: Yumi
authors.initials: Y
authors.lastname: Kanegae
mesh_terms: D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D002460:Cell Line; D005821:Genetic Techniques; D005822:Genetic Vectors / Q000302:isolation & purification; D006801:Humans; D019426:Integrases / Q000378:metabolism*; D010957:Plasmids; D014764:Viral Proteins / Q000378:metabolism*
chemical_list: D014764:Viral Proteins; C045073:Cre recombinase; D019426:Integrases