A Practical Guide for using the AdEasy System

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Tong-Chuan He, Kenneth W. Kinzler, and Bert Vogelstein Howard Hughes Medical Institute and Molecular Genetics Laboratory Johns Hopkins Oncology Center 424 North Bond Street Baltimore, MD 21231, USA Tel: (410)955-8886 Fax: (410)955-0548 vogelbe@welchlink.welch.jhu.edu kinzlke@welchlink.welch.jhu.edu t_c_he@yahoo.com

I. Introduction Summary Recombinant adenoviruses provide a versatile system for gene expression studies and therapeutic applications. We here report a strategy which simplifies the generation and production of such viruses. A recombinant adenoviral plasmid is generated with a minimum of enzymatic manipulations, employing homologous recombination in bacteria rather than in eucaryotic Cell s. Following transfections of such plasmids into a mammalian packaging cell line, viral production is conveniently followed with the aid of green fluorescent protein, encoded by a gene incorporated into the viral backbone. Homogeneous viruses can be obtained from this procedure without plaque purification. This system should expedite the process of generating and testing recombinant adenoviruses for a variety of purposes. Outline of the AdEasy system

Schematic Outline of the AdEasy System. The gene of interest is first cloned into a shuttle vector, e.g. pAdTrack-CMV. The resultant plasmid is linearized by digesting with restriction endonuclease Pme I, and subsequently cotransformed into E. coli. BJ5183 cells with an adenoviral backbone plasmid, e.g. pAdEasy-1,. Recombinants are selected for kanamycin resistance, and recombination confirmed by restriction endonuclease analyses. Finally, the linearized recombinant plasmid is transfected into adenovirus packaging cell lines, e.g. 293 cells. Recombinant adenoviruses are typically generated within 7 to 12 days. The "left arm" and "right arm" represent the regions mediating homologous recombination between the shuttle vector and the adenoviral backbone vector. An: polyadenylation site; Bm: BamHI, RI: EcoRI; LITR: left-hand ITR and packaging signal; RITR: right-hand ITR; Sp: SpeI.