Chapter 5 —— 131 —— PCR (jC and jT) and RFLP (×BmgBI) assays, respectively. Clones lacking AAVS1 targeted insertions or with only one HR-derived junction between transgenic and AAVS1 sequences are marked by red and orange arrowheads, respectively; whilst clones containing HRindependent donor DNA insertions are highlighted by yellow arrowheads. The remaining clones, modified through precise HR events involving the telomeric and centromeric side of the target sequence (jT and jC, respectively), are not highlighted. Clones with edited ATP1A1 alleles, scored via BmgBI amplicon digestion, are marked by green arrows. The cumulative datasets corresponding to these AAVS1 and ATP1A1 genotype screens (n=160 clones representing independent genome-modifying events), are plotted in Figure 1F. Supplementary Figure S3. Selector AAV gene editing of LMNA in hMSCs using combinatorial viral vector delivery. (A) Quantification of AAV gene editing with and without ouabain. hMSCs were co-transduced with AAV-HRLMN.A1 and AdVP.C9KARA at the specified doses. Gene editing frequencies were measured through mScarlet-directed flow cytometry at 3- and 24- days post-transduction (top and bottom graph, respectively). Prior to flow cytometry analysis at the latter timepoint, the hMSCs were cultured in the presence or absence of ouabain. Mock-transduced hMSCs and hMSCs transduced only with the highest dose of AAV-HRLMN.A1 provided for negative 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. (B) Characterization of LMNA protein tagging in hMSCs. LMNA tagging and nuclear localization was monitored by combining direct fluorescence microscopy for the reporter mScarlet and the DNA dye Hoechst 33342, respectively.
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