Chapter 4 —— 87 —— recorded at 2-h intervals for a period of 3 days using the software G/R Optical Module. Representative timelapses of experiments assessing the role of chromosomal DNA breaks on AAV vector transduction are available via: https://figshare.com/s/7efc29e8d0d34a99142a Tracking AAV donor knock-in kinetics HeLa cells were seeded at 8 × 104 cells per well of 24-well plates. The next day, the cells were transduced with AdVP.C9KARA (20 GC cell-1) together with AAV-HRS1 (0.5 TU cell-1) or with AAV-HRS1GS1 (0.5 TU cell-1). HeLa cells transduced exclusively with AAV-HRS1GS1 (0.5 TU cell-1) provided for controls. The tracing of AAV donor stable transduction levels in the presence and absence of targeted DSBs was performed by real-time fluorescence intensity measurements in an Incucyte S3 live-cell imaging analysis system (Sartorius) at 37℃ in a 5% CO2 atmosphere. Hence, expression of the livecell EGFP reporter served as a proxy for productive AAV transduction monitoring. Sixteen independent microscopy fields per well were recorded at 2-h intervals for a period of 3 days using the G/R Optical Module software. Representative timelapses of experiments monitoring AAV transduction kinetics under conditions favoring or not favoring gene knock-ins are available via https://figshare.com/s/3c09e1031a48e7df84c1 Statistical analyses The statistical parameters and analyses corresponding to the various experimental datasets are indicated in the respective figure legends whenever applicable. Acknowledgements The authors are thankful to Thilo M. Buck and Jan Wijnholds (Department of Ophthalmology, Leiden University Medical Centre, The Netherlands) for their advice during the setting-up of AAV vector production procedures in our laboratory and to Maarten van Dinther for his support with real-time livecell imaging experiments in the Incucyte live-cell imaging analysis system. The authors also thank the personnel of the Flow Cytometry Core Facility of
RkJQdWJsaXNoZXIy MTk4NDMw