Recombinase mediated cassette exchange into genomic targets using an adenovirus vector

David A. Sorrell, Claire J. Robinson, Jo-Ann Smith, Andreas F. Kolb

    Research output: Contribution to journalArticle

    16 Citations (Scopus)

    Abstract

    Recombinase mediated cassette exchange (RMCE) is a process in which site-specific recombinases exchange one gene cassette flanked by a pair of incompatible target sites for another cassette flanked by an identical pair of sites. Typically one cassette is present in the host genome, whereas the other gene cassette is introduced into the host cell by chemical or biological means. We show here that the frequency of cassette exchange is dependent on the relative and absolute quantities of the transgene cassette and the recombinase. We were able to successfully modify genomic targets not only by electroporation or chemically mediated gene transfer but also by using an adenovirus vector carrying both the transgene cassette to be inserted and the recombinase coding region. RMCE proceeds efficiently in cells in which the adenovirus vector is able to replicate. In contrast, insufficient quantities of the transgene cassette are produced in cells in which the virus cannot replicate. Additional transfection of the transgene cassette significantly enhances the RMCE frequency. This demonstrates that an RMCE system in the context of a viral vector allows the site directed insertion of a transgene into a defined genomic site.

    Original languageEnglish
    Article numbere123
    Number of pages12
    JournalNucleic Acids Research
    Volume38
    Issue number11
    Early online date5 Apr 2010
    DOIs
    Publication statusPublished - Jun 2010

    Keywords

    • embryonic stem-cells
    • site-specific recombination
    • mutant lox sites
    • CRE recombinase
    • transient expression
    • mammary-gland
    • gene
    • integration
    • protein
    • locus

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