Chapter 4 —— 74 —— incubated at room temperature (RT) for 15 min to allow for DNA-PEI complex formation. The formed complexes were then directly added in a dropwise fashion to the producer cells medium. At 6 h post-transfection, the transfection medium was replaced with fresh medium containing the E1deleted helper AdV vector AdV.SRα.LacZ.1.50 [27] at a multiplicity of infection (MOI) of 20 infectious units (IU) per cell for providing in trans all the proteins necessary for the replication and packaging of AdVP genomes into vector capsids. Importantly, AdV.SRα.LacZ.1.50 has its packaging signal flanked by loxP sites for selective Cre-mediated excision to hinder the assembly of helper vector particles. In addition, the PEC3.30 cells were transferred from 39℃ to 34℃ to allow for the proper folding of a thermosensitive version of the adenovirus DNA-binding protein expressed in PEC3.30 cells that further contributes to AdVP complementation. Upon the emergence of full cytopathic effect, the producer cells were collected and subjected to freezing and thawing for three cycles using liquid N2 and 37℃ water baths. After a centrifugation step at 2000 × g for 10 min, the clarified supernatants with rescued AdVPs were used for serial propagation in increasing numbers of producer cells co-transduced with the helper vector AdV.SRα.LacZ.1.50. For the rescue step and first two passages the helper was applied at 20 IU cell-1; whilst for the third final passage the helper was used at 40 IU cell-1. The AdVPs assembled at the last propagation round, involving 20 T175-cm2 culture flasks, were purified by sequential block and continuous CsCl buoyant density ultracentrifugation gradients. Finally, the retrieved AdVPs were de-salted by ultrafiltration through Amicon Ultra-15 100K MWCO filters (MerckMillipore, cat. no. UFC910024) against storage buffer A195 [28] consisting of 10 mM Tris (pH 7.4), 75 mM NaCl, 5% (w/v) sucrose, 0.02% Tween-80 (Sigma-Aldrich; cat. no.: BCBV8843), 1 mM MgCl2, 100 μm ethylenediaminetetraacetic acid (EDTA), 0.5% (v/v) ethanol and 10 mM histidine (Merck; cat. no.: 4350). The vector stocks were kept at –80℃ until usage.
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