Chapter 5 —— 125 —— transfection efficiency, all transfection mixtures were spiked with 80 ng of plasmid BE08_pCAG.mCherry.bGHpA expressing a red fluorescenceemitting reporter. After a 6-h incubation period, the medium of cells transfection-treated was removed, and the cells were transduced in regular culture medium containing AAV-HRS1.A1 particles at 1 TU cell-1. Ouabain selection of HeLa cells was initiated at 48 h post-trasdution with the concentration of 0.2 μM. HeLa cells were gone through flow cytometry at day 3 and day 20 to determine the transient and stable editing efficiencies, respectively, in the condition of ouabain selection or not. Testing selector AAV in-linkage donor HeLa cells were seeded 5 × 104 cells per well of 24-well plates. The next day, the cells were exposed or not to complexes formed by incubating 150 mM NaCl solutions containing 2.19 μl of PEI (1 mg ml-1) and plasmid mixtures consisting of 420 ng of AV62_pU.CAG.Cas9.rBGpA, AB65_pU.CAG.Cas9 D10A.rBGpA, or AB66_pU.CAG.dCas9.rBGpA as negative control. To control for transfection efficiency, all transfection mixtures were spiked with 80 ng of plasmid AZ15_pU.CAG.eGFP.rBGpA expressing a green fluorescence-emitting reporter. After a 6-h incubation period, the medium of cells transfection-treated was removed, and the cells were transduced in regular culture medium containing AAV-HRA1.IN17 particles at 1 TU cell-1. Ouabain selection of HeLa cells was initiated at day 4 post-trasdution with the concentration of 0.2 μM. HeLa cells were went through flow cytometry at day 3 and day 20 to determine the transient and stable editing efficiencies, respectively, in the condition of Ouabain selection or not. Viral vector transductions and DNA editing assays HeLa cells and hMSCs were seeded at, respectively, 2×104 and 5×104 cells per well of 48- and 24-well plates (Greinder Bio-One) and, after overnight incubation, they were mock-transduced or were transduced for 24 hours with combinations of AdVP.C9KARA (Li et al. 2024) and selector AAV vectors or with selector AAV vectors alone at the indicated MOIs. At 5- and 6-days post-
RkJQdWJsaXNoZXIy MTk4NDMw