P-MTAB-25970 - P-MTAB-25970
AAV8-CMV-eGFP and AAV8-CMV-hMIF vectors were produced in SF9 insect cells by using a baculovirus based system as described elsewhere. Briefly, SF9 suspension cultures were co-infected with three baculovirus stocks: 1) Bac-Rep containing the Rep gene from AAV2, 2) Bac-Cap8 containing the Cap gene from AAV8, 3) Bac-GFP, -hMIF harboring a CMV-cDNA expression cassette (eGFP and hMIF respectively) flanked by ITRs from AAV2. Each baculovirus stock was applied with an MOI of 3. 72 hours post infection the cellular fraction was collected by centrifugation. Cell pellets were resuspended and lysed by using a French Press. For removal of genomic DNA cell lysates were incubated with benzonase (50 U/mL) for 1 hour at 37C. Subsequently AAV particles were precipitated with CaCl2 (25 mM) followed by PEG precipitation (8% PEG-8000, 500 mM NaCl). After resuspension of PEG precipitates in 50 mM HEPES, 150 mM NaCl, 25 mM EDTA, pH 7.4 over night at 4C, AAV particles were further purified by CsCl density gradient centrifugation. Fractions from CsCl density gradients were analyzed by measuring the refractory index (RI) using a digital refractometer. Samples with a RI between 1.3774 and 1.3696 were pooled and dialyzed against PBS for removal of CsCl with a molecular weight cutoff of 20 kDa. Finally AAV preparations were further concentrated by using ultra filtration units. After addition of glycerol to a final concentration of 10%, AAV preparations were sterile filtered, aliquoted, frozen and subsequently stored at -80C.Genomic titers of purified AAV stocks were determined by isolation of vector-DNA and subsequent qPCR analysis using CMV-promoter specific primers.