The CreloxP System for Generating HDAds

The first efficient and currently most widely used method for generating HDAd was the Cre/loxP system developed by Graham and coworkers in 1996 (54) (Fig. 2). In most versions of this system, the HDAd genome is first constructed in a bacterial plasmid. Minimally, the HDAd genome contains the expression cassette of interest and ~500 bp of cis-acting Ad sequences necessary for vector DNA replication (ITRs) and packaging (^). As described in detail in Section V.E, inclusion of stuffer DNA in the HDAd genome is often required for efficient packaging. To rescue the HDAd (i.e., to convert the ''plasmid form'' of the HDAd genome into the ''viral form''), the plasmid is first digested with the appropriate restriction enzyme to liberate the HDAd genome from the bacterial plasmid sequences. 293 Cells expressing Cre are then transfected with the linearized HDAd genome and subsequently infected with the helper virus. The helper virus bears a packaging signal flanked by loxP sites and thus, following infection of 293Cre cells, the packaging signal is excised from the helper viral genome by Cre-mediated site-specific recombination between the loxP sites. This renders the helper viral genome unpackagable, but still able to undergo DNA replication and thus trans-complement the replication and encapsida-tion of the HDAd genome. The titer of the HDAd is increased by serial coinfection of 293Cre cells with the HDAd and the helper virus (Fig. 3). Maximum HDAd titer of ~ 108 infectious particles/mL (~ 1000-2000 infectious particles/producer cell) is usually obtained by the third serial passage (Fig. 3)(55). Typically, the level of helper virus contamination is (<1%, as determined by Southern blot hybridization analysis, before vector purification by CsCl ultracentrifugation (55) and, provided that the genome sizes of the vector and helper are sufficiently different, purities of <0.1 % helper virus contamination can be achieved following CsCl ultracentrifugation as determined by Southern blot hybridization or quantitative real-time polymerase chain reaction (PCR) (56). Detailed methodologies for producing HDAd using the Cre/loxP system are described elsewhere (57). A similar system was subsequently reported by Hardy et al. (58).

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