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).