Chapter 4 —— 77 —— (pH 7.4), subjecting the resuspended cells to three cycles of freezing and thawing in liquid N2 and 37℃ water baths, respectively, and finally removing the resulting cell debris by centrifugation at 3220 × g for 15 min at 4℃. The 17-ml AAV vector suspensions were subsequently treated with 50 U ml-1 of Benzonase (Millipore; cat. no.: E1014-25KU) for 1 h at 37℃ and then centrifuged at 2420 × g for 10 min at 4℃. Next, clarified supernatants containing AAV particles were loaded onto Iodixanol-OptiPrep (Progen; cat. no.: 1114542) cushions of 15%, 25%, 40% and 60% placed in Quick-Seal round-top polypropylene tubes (Beckman; cat. no.: 342414). The AAV particles were then purified and concentrated via iodixanol gradient ultracentrifugation at 69 000 revolutions per minute in a 70Ti rotor (Beckman Coulter) at 16℃ in a Beckman Coulter Optima XE-90 centrifuge. By piercing the ultracentrifuge tubes with a needle (18G needle BD MicrolanceTM; cat. no.: 304622), the majority of AAV particles located within the 40% iodixanol cushion, were collected and subjected to buffer exchange using Amicon Ultra-15 100K MWCO filters (Millipore; cat. no.: UFC910024) and Dulbecco’s phosphate-buffered saline (Thermo Fisher Scientific; cat. no.: 14040–091) containing 0.001% Poloxamer 188 (Sigma-Aldrich; cat. no.: P5556). Purified AAV batches were stored at –80℃ and the respective transducing unit (TU) titers were determined by limiting dilution assays on HeLa cells as follows. Firstly, 5 × 104 cells were seeded in wells of 24-well plates (Greiner Bio-One) and 16–18 h later, the cells were exposed to 3-fold serial dilutions of each vector preparation. At 3 days post-transduction, the frequencies of transduced cells were determined by flow cytometry with the corresponding functional AAV vector titers corresponding to HeLa-cell TU per ml being calculated as the percentage of transduced cells × cells seeded × dilution factor x 1000/μl. The AAV vector batches produced, and their respective titers are listed in Supplementary Table S1. The transducing GC titers of AAV-HRLMNAGEX1 were established via qPCR assays using the iQ SYBR Green Supermix (Bio-Rad, cat. no. L010171C)
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