Production And Characterization

Because HDAds are deleted of all viral coding sequences, a helper virus is required for their propagation. The first efficient and currently most widely used method for generating HDAd is the Cre/loxP system developed by Graham and coworkers in 1996[2] (Fig. 1). In 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 (C). Because efficient packaging into the Ad capsid requires a genome size between ~27.7 and ~ 38 kb, ''stuffer'' DNA is usually included in HDAd. To rescue the HDAd (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 the site-specific recombinase 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, the target sequence for Cre, 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 encapsidation of the HDAd genome. The titer of the HDAd is increased by serial coinfection of 293Cre cells with the HDAd and the helper virus and the HDAd is finally purified by CsCl ultracentrifugation. Subsequent improvements of this original system permit efficient and simple large-scale HDAd production with yields of > 10,000 vector particles per coinfected producer cell and with helper virus

Fig. 1 The Cre/loxP system for generating HD vectors. The HDAd contains only ~ 500 bp of cis-acting Ad sequences required for DNA replication (ITRs) and packaging (C), the remainder of the genome consists of the desired transgene and non-Ad ''stuffer'' sequences. The HDAd genome is constructed as a bacterial plasmid (pHDAd) and is liberated by restriction enzyme digestion (e.g., PmeI). To rescue the HDAd, the liberated genome is transfected into 293Cre cells and infected with a helper virus bearing a packaging signal (C) flanked by loxP sites. Cre-mediated excision of C renders the helper virus genome unpackagable, but still able to replicate and provide all of the necessary trans-acting factors for propagation of the HDAd. The titer of the HD vector is increased by serial coinfections of 293Cre cells with the HDAd and the helper virus. (View this art in color at www.dekker.com.)

contamination levels of <0.02%.[3] Detailed methodologies for producing HDAd are described in detail elsewhere.[3'4]

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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