Chapter 4 —— 86 —— 30-s. A melting curve was generated by subjecting the samples to 95℃ for 10 s and subsequently to 65℃ to 95℃ with 5-s 0.5℃ increments. The relative amounts of AAV vector genomes were expressed in PCR Ct values corresponding to individual transduction conditions. Tracking of AAV versus scAAV transduction kinetics HeLa cells were seeded at 8 × 104 cells per well of 24-well plates. The next day, the cells were transduced with AAV-HRS1 or scAAV-HRS1 at 0.5 TU cell-1 and placed in an Incucyte S3 live-cell imaging analysis system (Sartorius) at 37℃ in a 5% CO2 atmosphere. Real-time fluorescence intensity measurements derived from 16 independent microscopy fields per well were recorded at 2-h intervals for a period of 3 days using the software G/R Optical Module. Representative timelapses of experiments establishing faster transduction kinetics of scAAV over regular AAV vectors are available via: https://figshare.com/s/8e3e6c8ef5438d9c3064 Tracking AAV vector transduction in cells with chromosomal breaks HeLa cells were seeded at 8 × 104 cells per well of 24-well plates. The next day, the cells were exposed to complexes formed by incubating 150 mM NaCl solutions containing 1.54 μl of PEI (1 mg ml-1) and plasmid mixtures consisting of 300 ng of AV62_pCAG.Cas9.rBGpA [37] and 100 ng AG66_pgRNACALM2 [36] or 300 ng of AV62_pCAG.Cas9.rBGpA and 100 ng of AG65_pgRNAVEGFA [36] or, as negative control, 300 ng of AV62_pCAG.Cas9.rBGpA and 100 ng of pMoluc (Addgene plasmid #1251). To control for transfection efficiency, all transfection mixtures were spiked with 50 ng of plasmid BE08_pCAG.mCherry.bGHpA expressing a red fluorescence-emitting reporter. After a 6-h incubation period, the transfection medium was removed, and the cells were transduced in regular culture medium containing AAV-HRS1 or scAAV-HRS1 at 0.5 TU cell-1. The timedependent AAV and scAAV transduction levels were followed in an Incucyte apparatus at 37℃ in a 5% CO2 atmosphere. Real-time fluorescence intensity measurements derived from 16 independent microscopy fields per well were
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