Development of novel method for the construction of viral vectors using the site-specific recombinase

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Recently, many kind of novel gene have been cloned during genome project, however the characterization of these genes was not sufficient. The virus vector can be a candidate for this efficient characterization, however construction of numerous virus vectors at once, as an expression library, has been difficult. In this research, we demonstrated a unique system for generating viral vectors as a library by directly substituting a gene of interest in a small-transfected plasmid (donor plasmid) with a replaced gene in a replicating viral genome in the site specific recombinase-expressing cells using the recombinase-mediated cassette exchange (RMCE) reaction. For example, we chose the replication-deficient adenovirus vector (1st generation rAd) for the virus vector and Cre/loxP system for RMCE. We have already determined the exclusive mutants loxP (V), which does not recombine with wild type loxP (wt loxP) but efficiently only with V. In combination with a positive selection of the viral ci … More s-acting packaging signal connected with the insert, the purpose vector was enriched to over 90% after four cycles of infection in Cre-expressing 293 cells. And this RMCE reaction was able to substitute DNAs even when wt loxP and V are 30kb apart, though this efficiency decreased.On the other hand, the fidelity of RMCE was examined using a donor plasmid lacking V. After three cycles of infection in Cre-expressing 293 cells, the GFP-expressing rAd, which was used for the gene of interest in this research and was expected to be generated only after correct RMCE, was detected. We isolated these unexpected rAds by limiting dilution and characterized the genome structures using restriction-enzyme digestion and sequencing. We detected many kind of non-homologous recombination products generated during the RMCE in these unexpected rAds. However, we may control generation of this unexpected rAd production by optimizing of the efficiency of RMCE and minimizing the number of cycles infection.Because this novel method to construct the virus vector using RMCE reaction was efficiently generating the purpose vector, this method may also be applicable to not only for the development of the other DNA virus vector but also for viral-based cDNA-library. Less