Chapter 5 —— 108 —— with the highest and lowest AAV-HRS1.A1 dosages, respectively (Figure 2C). Junction PCR and RFLP analyses (Figure 2A) of genomic DNA from stably transduced cell populations established ouabain-dependent co-selection of cells edited at AAVS1 and ATP1A1 (Figure 2D). Figure 2. Selector AAV gene targeting at AAVS1 and ATP1A1 using combinatorial viral vector delivery. (A) Schematics of the experimental setup. Selector AAV-based gene targeting upon co-transduction of HeLa cells with vector AAV-HRS1.A1, encoding donor and gRNA sequences, and vector AdVP.C9KARA, encoding a Cas9 nuclease, was assessed via EGFP-directed flow cytometry and genotyping assays based on junction PCR and RFLP assays as depicted. (B) Quantification of selector AAV donor delivery. HeLa cells were transduced with AAV-HRS1.A1 alone or together with AdVP.C9KARA at the specified multiplicities of infection (MOI). Transduction levels were determined by flow cytometric quantification of EGFP-positive cell frequencies and respective mean fluorescence intensity (MFI) values at 3 days post-transduction (left and right graphs, respectively). Mock-transduced cells provided for negative controls. (C) Quantification of selector AAV-based DNA editing with and without ouabain. AAV stable transduction frequencies were measured via EGFP-directed flow cytometry after sub-culturing HeLa cells initially exposed to the indicated vector doses for 20 days. During sub-culturing, the cells were incubated or not with ouabain. Mock-transduced cells and cells transduced exclusively with AAV-HRS1.A1 served as controls. The results are presented as mean ± SD of three biological replicates. Significant differences amongst the marked datasets were calculated by Student’s t-tests; **P<0.05. (D) Genotyping of AAVS1 and ATP1A1 in co-
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