Characterization of recombinant baculovirus
The identity of the baculoviral genomes was verified by Sanger sequencing from PCR products of DNA extracts. The copy numbers of the AAV genome and RepCap cassette were determined after DNA extraction using a High Pure viral nucleic acid kit (Roche Life Science) followed by a qPCR using the sets of primers and probes described previously (Penaud-Budloo et al., 2017).
For the stability study of the baculovirus clones over passages, 2ml of Sf9 cells were seeded per well at a density of 1x106cells per ml in a 6-wells plate. For each clone, cells were infected with the baculovirus supernatant of either of the passages P1 to P10 at multiplicity of infection (MOI) of 1 infectious unit as determined by CSA assay and incubated for 72 hours at 27°C in a humidified chamber. After low speed centrifugation 5 min at 500 x g, the cells pellet was recovered at each passage in order to verify AAV Rep and Cap proteins expression by SDS-PAGE and western blotting. Total proteins were extracted in RIPA buffer supplemented with 1X protease inhibitor cocktail (Sigma-Aldrich, MO) and protein concentration was determined using the kit DC protein assay (Bio-Rad, CA). Five micrograms of total proteins were loaded on a 10% Tris-Glycine mini-gel (ThermoFisher Scientific, MA) and run at 1ml/cell, 100V for 2.5 hours. Proteins were transferred onto a PVDF membrane (ThermoFisher Scientific, MA) for 7 min at 25V, 1.3 A constant, using a Trans-Blot Turbo Transfer System (Bio-Rad, CA). Membranes were incubated overnight at 4°C in blocking buffer (1X PBS, 0.1% Tween 20, 5% milk powder) and 1h at room temperature in the primary antibody solution (1X PBS, 0.1% Tween 20, 0.5% milk powder, primary antibody). The Rep303.9 antibody or the anti-AAV VP1/V2/VP3 B1 antibody (Progen, Heidelberg, Germany) are used at a dilution 1:20 or 1:2,000 for the detection of Rep and Cap proteins, respectively. Membranes were washed three times for 10 min in the washing buffer (1X PBS, 0.1% Tween 20) and incubated for 1h at room temperature with the secondary antibody HRP-linked goat anti-mouse diluted at 1:2,000 (Agilent Dako, CA). After three additional washes for 10 min, the chemiluminescent signal was revealed using the Western Pierce ECL substrate (ThermoFisher Scientific, MA) and exposure with Amersham Hyperfilm ECL (Cytiva GE Life Sciences, MA).