293FT cells were counted using a Vi-Cell XR Cell Counter (Beckman Coulter) and 1 × 107 cells were plated using DMEM media containing FBS without penicillin and streptomycin on a 15-cm tissue-culture dish. The following day, the cells were transfected using Lipofectamine 2000 with the AAV vector along with AAV-DJ and AAV-pHelper packaging vectors (Cell Biolabs) at a ratio of 1:1:1. The next day, fresh DMEM media with 10% FBS was added; 48 h later, the cells and media were collected and subjected to four freeze-thaw cycles by alternating between an ethanol dry ice bath and 37°C. Cell debris was removed by centrifugation and the supernatant was collected, passed through a 0.45-µm filter, aliquoted, and frozen at −80°C until use.
To generate high-titer AAV for in vivo experiments, AAV was generated as described above, but 10 15-cm tissue-culture dishes of 293FT cells were transfected with AAV, and all transfected cells were pooled when the virus was harvested. AAV virus was purified using Virabind AAV Purification Megakit (Cell Biolabs #VPK141) according to the manufacturer's instructions. AAV virus was titered using QuickTiter AAV Quantitation Kit (Cell Biolabs ##VPK145) according to the manufacturer's instructions.
To generate high-titer AAV for in vivo experiments, AAV was generated as described above, but 10 15-cm tissue-culture dishes of 293FT cells were transfected with AAV, and all transfected cells were pooled when the virus was harvested. AAV virus was purified using Virabind AAV Purification Megakit (Cell Biolabs #VPK141) according to the manufacturer's instructions. AAV virus was titered using QuickTiter AAV Quantitation Kit (Cell Biolabs ##VPK145) according to the manufacturer's instructions.