Peipro

293T cells (ECACC 12022001, RRID:CVCL_0063) were grown on multi-well plates in high-glucose Dulbecco’s Modified Eagle Medium (DMEM, Gibco 11965084) with 10% fetal bovine serum (FBS, Thermo Fisher 10091-148) and 1% Penicillin-Streptomycin (Gibco 15070-063). Cells were seeded a day prior to transfection or transduction. For transfections, 286 ng of plasmid was used per square centimeter of well area. PEIpro (Polyplus Transfection, Illkirch, France, 115-100) was mixed in DMEM with plasmid at 1 μg of PEIpro for 1 μg of plasmid DNA, and then incubated at room temperature for 15 min before adding to the cells. In transduction experiments, the vectors were used at 20,000 MOI (multiplicity of infection), diluted to an appropriate volume with DMEM. To enhance the transduction, an hour prior to it, Compound C (7.5 μM, Sigma-Aldrich, Saint Louis, MI, USA, 171261) was added to the cells (33 (link)).
Inducer (depending on the riboswitch, tetracycline, or toyocamycin) was added to cells either directly or 24 hpt (hours post-transfection). Both inducers were used from a stock solution diluted in sterile water, at a 10 mg/mL concentration for tetracycline and 1 mg/mL for toyocamycin. The final concentrations used in cell culture were 5 μM for toyocamycin, and varying, but usually 100 μM for tetracycline. Cells were generally collected at 48 hpt.

AAV9-ITS01 vectors were produced by triple transfection in HEK293T cells. HEK293T cells were split the day before transfection to reach a 60-80% confluency when transfected. Expression plasmids encoding Rep2/Cap9, pHelper, and pAAV.CASI.ITS01 were mixed in serum-free OptiMEM at a 1:1:1 ratio at a total of 60 µg DNA per flask being transfected. Transfections were conducted using PEIpro following the manufacturer’s instructions (Polyplus Transfection). At 3 days post transfection, cells were harvested by adding 0.5M EDTA, pH 8.0 to each flask. Cells and medium were centrifuged at 2000×g for 10 minutes. Cells were washed twice with PBS and then lysed using an AAV lysis buffer (150mM NaCl, 2mM MgCl2, 50mM Tris-HCl pH 8.0). Cells in lysis buffer were subjected to three freeze/thaw cycles. Benzonase (50U/mL) and Triton X-100 (0.01%) were added to the lysate mixture and the mixture was incubated for 1 hour at 37°C. The lysate mixture was centrifuged at 7000×g for 1 hour, the supernatant was collected and filter sterilized with a 0.45 µM filter. POROS Capture Select AAV9 columns were used to purify AAV9 vectors. Eluted vectors were buffer exchanged into sterile PBS and concentrated using Amicon Ultra Centricon Tubes. Vectors were quantified by qPCR with a Roche Lightcylcer 480ii in parallel.