Chapter 4 —— 75 —— The AdVP transducing genome copy (GC) titers were established via quantitative PCR (qPCR) assays using a previously published protocol [25] based on the iQ SYBR Green Supermix (Bio-Rad, cat. no. L010171C) and a primer pair specific for the AdV packaging signal. (i.e.5′- CGGTGTACACAGGAAGTGACA-3′ and 5′-CAGATTTCACTTCCTCTT ATTCAG-3′). In brief, HeLa cells seeded one day before at a density of 8 × 104 cells per well of 24-well plates (Greinder Bio-One), were exposed to seven 3-fold serial dilutions of each purified AdVP stock. Genomic DNA extracted at 24 h post-transduction with the DNeasy Blood & Tissue kit (QIAGEN; cat. no.: 69506) was then subjected to qPCR analysis. In parallel, eight serial 10-fold dilutions of linearized parental AdVP plasmid stocks containing 1 × 107 GC ml-1 were used to generate a qPCR standard curve. Data analysis was carried out by using the Bio-Rad CFX Manager 3.1 software (Bio-Rad Laboratories) with the titers being subsequently calculated on the basis of the cycle threshold (Ct) values corresponding to the AdVP genome dilutions and plasmid DNA standard curves [25]. The structural integrity of AdVP genomes was established by restriction fragment length analysis assays as follows. First, 50 μl of purified AdVPs were incubated with 8 μl of 10 mg ml-1 DNase I (Sigma-Aldrich, cat. no. 10104159001) for 30 min at 37℃ and, subsequently, the DNase I activity was inactivated by adding 1.5 μl of 20 mg ml-1 proteinase K (Thermo Fisher Scientific, cat. no. EO0491), 6 μl of 10% (w/v) sodium dodecyl sulfate (SDS) and 2.4 μl of 0.5 M EDTA (pH 8.0). After a 1-h incubation period at 55℃, AdVP genomes were isolated by using the QIAEX II Gel Extraction Kit (QIAGEN, cat. no. 20021) according to the manufacturer’s recommendations. The isolated AdVP genomes were then subjected to the indicated restriction enzyme digestions and analyzed after agarose gel electrophoresis by using the Gel-Doc XR + system (Bio-Rad Laboratories) and the Image Lab 6.0.1 software (Bio-Rad Laboratories). The AdVP parental plasmids U67_pHCAdVP.C9KARA and S90_pHC-AdVP.eC94NLSGS1 digested with the same restriction enzymes applied to AdVP genomes provided for molecular weight
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