Chapter 5 —— 116 —— Figure 5. Selector in-linkage AAV gene targeting using combinatorial viral vector delivery. (A) Diagram of the experimental setup. The selector AAV-HRA1.IN17 vector contains a gRNA unit specific for intron 17 of ATP1A1 and a matched HR donor with the ouabain-resistance polymorphism T804N in its ATP1A1 homologous region. LHA and RHA, “left” and “right” homology arms, respectively. (B and C) Quantification of selector AAV donor delivery. hMSCs and HeLa cells (panel B and C, respectively) were transduced with AAV-HRA1.IN17 alone or together with AdVP.C9KARA at the specified MOI. Transduction levels were determined by flow cytometric quantification of mScarletpositive cell frequencies and respective MFI values at 3 days post-transduction (left and right graphs, respectively). Mock-transduced cells provided for negative controls. (D and E) Quantification of selector in-linkage AAV gene targeting with and without ouabain. Gene targeting frequencies in hMSC and HeLa cell cultures (panel D and E, respectively) were measured via mScarlet-directed flow cytometry after sub-culturing these cultures initially exposed to the indicated vector doses in the presence and absence of 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.
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