Development of new method for construction of adenovirus vector using gene exchange reaction

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To develop a method for constructing adenovirus vector using gene exchange reaction mediated by Cre recombination. We assayed mutant loxPs recognized independently of wild-type loxP, authentic target sequences of Cre, and identified loxP V as a mutant loxP with high sensitivity and specificity. We examined efficiency of gene exchange reaction using both wild-type loxP and mutant loxP V and showed that the efficiency was no less than 10 %.The recipient virus for constructing recombinant adenovirus of El -deleted type was designed so that the viral packaging signal (Ψ) was able to be excised by Cre-mediated recombination between a pair of loxP sites located at the both sides of Ψ sequences. We introduced to Cre-expressing 293 cells both a recipient virus and a donor plasmid containing Ψ sequences and a purpose gene flanked with loxP and V. After five cycles of infection of Cre-293 cells with crude viral stock obtained, an adenovirus vector expressing the purpose gene was generated through gene exchange reaction. Pure purpose virus vector was able to be easily isolated after limited dilution using 293 cells.To construct "gutted vector" whose all viral genes were replaced by a purpose DNA, we prepared a new recipient virus that serves as a helper virus. We also constructed four different donor cosmid cassettes containing cleavage sites of 8-base recognition enzyme as a cloning site in order to insert a purpose gene and a stuffer DNA of large size.