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.