Construction of first-generation adenoviral vectors.

Genetically modified adenoviruses (Ads) make attractive vectors for the delivery of exogenous DNA to mammalian cells for basic science and gene therapy applications. Ad vector production consists of (1) cloning a trangene into an infectious plasmid by in vivo recombination in bacteria, (2) rescuing and propagating the vector in complementing cells, and (3) purifying the vector. All of this can be accomplished using commercially available reagents, plasmids, and cell lines. Helper-independent Ads have a large cloning capacity (5–14 kbp) and transduce efficiently in a wide range of both quiescent and proliferating cell types. They propagate readily and produce high-titer stocks (1011 to 1013 vector particles from a 3-liter culture). Furthermore, Ads rarely integrate into the host genome and are relatively safe. However, Ad vector production typically takes 4–6 weeks, and promiscuous host-cell transduction can occur in vivo. Furthermore, immune responses against viral proteins encoded by the vector backbone can occur, and transgene expression by Ad vectors is relatively short. Here, we discuss the current methods for the generation, propagation, purification, and characterization of helper-independent Ad vectors. INTRODUCTION, ### PROTOCOL, ### Construction and Characterization of Ad Vectors, ### MATERIALS, ### Reagents, ### Equipment, ### METHODS, ### Generation of Infectious Ad Plasmids, ### Cell Preparation, ### Transfection for Rescue of Ad Vectors, ### Analysis and Expansion of Plaque-isolated Ad Vector, ### Large-scale Preparation of Ad Vectors, ### Vector Purification, ### Characterization of Purified Ad Vectors, ### ACKNOWLEDGMENTS, ### REFERENCES, ### 15 Construction of First-generation Adenoviral Vectors P. Joel Ross*† and Robin J. Parks*†‡ *Molecular Medicine Program, Ottawa Health Research Institute, Ottawa, Ontario, Canada; †Department of Biochemistry, Microbiology, and Immunology, Centre for Neuromuscular Disease, and ‡Department of Medicine, University of Ottawa, Ottawa, Ontario, Canada