Paav dj plasmid

AAV vectors were prepared and tittered as described previously, with some slight modifications [15 (link)]. Briefly, AAV plasmid, pHelper plasmid (Cell Biolabs, San Diego, CA, USA), and pAAV-DJ plasmid (Cell Biolabs, San Diego, CA, USA) were transfected into HEK293FT cells, and 72 h later, HEK293FT cells were harvested. AAVs were extracted through four freeze-thaw cycles and purified by two Cscl/PBS ultracentrifugations using a SWi40 rotor. After the 2nd round of ultracentrifugation, AAV-rich fraction was collected and dialyzed using a dialysis cassette Slide-A-lyzer (#87730, Thermo Scientific, IL, USA) to remove Cscl. qPCR (#QKD-201, TOYOBO, Osaka, Japan) was performed to estimate final AAV titer.

AAV vectors were prepared and titered as described previously [25 (link)]. AAV plasmid, pHelper plasmid (Cell BioLabs), and pAAV-DJ plasmid (Cell BioLabs) were cotransfected into 293AAV cells. Seventy-two hours after transfection, cells were harvested. AAV vectors produced in 293AAV cells were purified by CsCl gradient ultracentrifugation using a SW41Ti rotor (Beckman-Coulter Life Science, Indianapolis, IN). The formed gradients were fractionated, and the AAV titer of each fraction was evaluated by qPCR (TOYOBO, Osaka, Japan). AAV-containing fractions were combined and subjected to a second round of CsCl gradient ultracentrifugation for further purification. After ultracentrifugation, the AAV titer of each fraction was evaluated by qPCR. AAV-rich fractions were combined and dialyzed using a dialysis cassette Slide-A-Lyzer (Thermo Fisher Scientific) to remove CsCl. The final AAV titer was determined by qPCR.