Chapter 4 —— 76 —— references. The in-silico restriction patterns corresponding to vector and reference plasmid DNA were generated by using the SnapGene software (version 6.0.7). AAV vector production, purification and characterization The AAV vectors were generated by transfecting HEK293T cells seeded one day before in T175-cm2 culture flasks at a density of 2 × 107 cells per flask (up to 18 flasks per AAV stock). The transfected DNA consisted of each AAV transfer plasmid (Supplementary information) and the packaging plasmid AT51_pDG6.RSV.DsRed.SV40pA mixed at 1:1 molar ratio (30 μg total DNA per flask). As aforementioned, this packaging construct expresses AAV serotype-2 rep and AAV serotype-6 cap together with adenovirus helper functions [23]. For each T175-cm2, mixtures consisting of DNA and 99 μl of PEI (1 mg ml-1) each diluted in 1 ml of 150 mM NaCl. Transfection mixtures were generated by dropwise addition of the PEI to the DNA solution followed by immediate homogenization in a vortex for 10 s. After incubation at room temperature for 16–18 min, the formed DNA-PEI complexes were directly added to the HEK293T cells and, 24 h later, the transfection medium was replaced by 20 ml of fresh medium. At 5 days post-transfection, the producer cells were detached with a cell scrapper and transferred together with the conditioned medium into 50-ml tubes and then centrifuged at 1000 × g for 10 min at 4℃. The resulting supernatants and cell pellets were separately recovered and stored at –80℃ overnight. After thawing, each 100 ml of supernatant received 25 ml of a 40% (w/v) poly(ethylene) glycol 8000 solution (PEG 8000; Sigma-Aldrich; cat. no.: P2139) with the resulting mixture being subsequently slowly stirred for 1 h at 4℃ followed by an overnight incubation at 4℃ without stirring for full precipitation. Next, the supernatant-PEG 8000 mixtures were centrifuged at 2820 × g for 15 min at 4℃ in 50-ml tubes and the resulting pellets were resuspended in 7 ml of phosphate-buffered saline (PBS) (pH 7.4) and mixed with 10 ml of clarified cell lysates to yield 17 ml of vector suspensions. The clarified cell lysates were obtained after resuspending the producer cell pellets in 10 ml of PBS
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