Use of the Cosmid Adenoviral Vector Cloning System for theIn VitroConstruction of Recombinant Adenoviral Vectors

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Abstract
The large size of the adenoviral genome unfortunately precludes there being many unique, useful restriction sites available for in vitro manipulation. Two methods have been developed for the construction of recombinant adenoviral vectors to date: in vivo homologous recombination or direct ligation in vitro. The efficiency of either the direct ligation method or the homologous recombination method is low because of the large size of the recombinant adenoviral vectors. To circumvent these problems, we have chosen to use the cosmid vector system to facilitate the assembly of recombinant adenoviral vectors. In this paper, we demonstrate for the first time that recombinant adenoviral vectors can be efficiently constructed in vitro by the cosmid vector system. With this method, it is possible to amplify the recombinant adenoviral vector DNA sufficiently to transfect 293 cells. The cosmid adenoviral vector cloning method for in vitro construction of the full-length recombinant adenoviral vectors represented here is simple and efficient and should facilitate the development of recombinant adenoviral vectors for human gene therapy. Construction of recombinant adenoviral vectors requires much work. In this paper, we describe the cosmid adenoviral vector cloning system that will facilitate development of recombinant adenoviral vectors. We first used a three-way ligation to introduce cosmid cohesive ends into a full-length recombinant cosmid adenoviral cloning vector. The two fragments generated from the recombinant cosmid adenoviral cloning vector can be used to construct, using a two-way ligation, a recombinant adenoviral vector with much less work. The cloning capacity in this system is up to 8 kb so far. Because of the high efficiency of the cosmid cloning system and lack of wild-type adenovirus contamination, the cosmid adenoviral vector cloning method is simple and efficient to develop recombinant adenoviral vectors for human gene therapy.