Protocol

Construction and Characterization of Adenovirus Vectors

Adapted from Gene Transfer: Delivery and Expression of DNA and RNA (eds. Friedmann and Rossi). CSHL Press, Cold Spring Harbor, NY, USA, 2007.

INTRODUCTION

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 transgene 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. First-generation Ads have a large cloning capacity (5-14 kbp) and efficiently transduce a wide range of both quiescent and proliferating cell types. They are readily propagated to produce high-titer stocks (10¹¹-10¹³ vector particles from a 3-L culture). Furthermore, Ads rarely integrate into the host genome and are relatively safe. However, Ad vector production typically takes 4-6 wk, and promiscuous host-cell transduction can occur in vivo. Furthermore, immune responses against viral proteins encoded by the vector backbone can occur, which limits the duration of transgene expression in vivo. Regardless of these limitations, Ad remains one of the more versatile and efficient gene delivery systems. Here, we discuss methods for the generation, propagation, purification, and characterization of first-generation Ad vectors.