[2] Techniques for human adenovirus vector construction and characterization
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This chapter describes the techniques for human adenovirus vector construction and characterization. Human adenoviruses (Ads) have attracted considerable attention lately for their potential use as vectors for gene therapy, for recombinant vaccines, and for high-level protein expression in mammalian cells. Among the reasons for these interests are the following: (1) the 36 kilobase pair (kbp) double-stranded DNA genome of Ad is relatively easy to manipulate by recombinant DNA techniques and (2) Ad can infect and direct high levels of protein expression in both proliferating and quiescent cells, an important feature for vectors requiring in vivo expression. The chapter describes several plasmid-based systems for inserting foreign genes into the Ad genome and the methods used to purify, grow, and titrate recombinant viruses. The vectors are based on the human Ad5 genome, the structure of which is presented in the chapter. In a normal infection, early genes (E1A, EIB, E2, E3, and E4) are expressed prior to DNA replication. Late gene expression, driven predominantly by the major late promoter at 16 map units, occurs after DNA replication. The products of the EIA gene are required for the expression of all the other Ad genes. Thus, E1 deletion viruses are defective for replication in all cell types except the E1-complementing 293 cell line. The E3 region, which may be important for virus persistence in vivo, is dispensable for growth of the virus in vitro. Both the E1 and E3 regions, therefore, provide convenient sites for insertions of foreign sequences to generate helper-independent recombinant viruses.
