rAAV characterization
For real-time PCR analyses, 3µl of each purified rAAV stock was
pretreated or not with 20U of DNase I (Roche, Basel, Switzerland) before
DNA extraction in a total volume of 200µl of DNase reaction buffer (13
mM Tris pH 7.5, 0.12 mM CaCl2, 5 mM
MgCl2) for 45 min at 37°C. The vector genome (vg) copy
number was determined after DNA extraction, using a High Pure viral
nucleic acid kit (Roche Life Science). Baculoviral DNA contamination was
quantified by qPCR assays targeting resistance genes kanamycin
(“kana”) using the set of primers Kana-F 5’- GGGCGCCCGGTTCTTTTTGTC,
Kana-R 5’- GCCAGTCCCTTCCCGCTTCAGTG and Kana-Pr 5’- CCGACCTGTCCGGTGCCCTG
and the gentamycin (“genta”) using the set of primers Genta-F 5’-
AGCCCGCATGGATTTGAC, Genta-R 5’- GGGCATCATTCGCACATGTA and Genta-Pr 5’-
TGGTCAGGGCCGAGC. The copy numbers of the rep-cap sequences and the Sf9
genome were determined by quantification of the rep2 gene and Sf
ribosomal protein L37A gene amplicons, respectively, as previously
reported (Penaud-Budloo et al., 2017).
For rAAV vector characterization, SDS-PAGE was performed from 2.5 to
5x109 vector genomes and western blotting was realized
as described above for the detection of AAV Cap proteins. Vector purity
was also evaluated by SDS-PAGE followed by silver staining (PlusOne
silver stain kit; GE Healthcare Life Sciences) of
2.1010 vector genomes of each rAAV stock.