Chapter 4 —— 82 —— reaction (RT-PCR) analysis targeting endogenous and tagged DMD transcripts. The capacity of mock- and AdVP-transduced hMSCs to differentiate into osteoblasts was tested as follows. After seeding hMSCs at 5 × 104 cells per well of 24-well plates, the cells were either treated or not treated with AdVP.C9KARA at 2 TU cell-1. After a 10-day sub-culturing period, the hMSCs were seeded at 1 × 104 cells in wells of 48-well plates and cultured for 2 weeks in osteogenic differentiation medium consisting of MEM-α containing 10% FBS, 100 U ml-1 penicillin/streptomycin, 1 × non-essencial amino acids, 1 × GlutaMax, 0.2 mM l-ascorbic acid 2-phosphate (Sigma-Aldrich; cat. no.: A8960), 10 mM β-glycerophosphate (Sigma-Aldrich; cat. no.: G6251), 2 μM dexamethasone (Sigma-Aldrich; cat. no.: D4902) and 100 ng ml-1 of recombinant human bone morphogenetic protein 6 (BMP6; PeproTech; cat. no.: 120–06). The differentiation medium was refreshed every 3–4 days and alizarin red S staining was performed for detecting calcium deposits. In brief, after fixing hMSCs in 4% paraformaldehyde in phosphate-buffered saline (PBS; pH 7.4) for 10 min, the cells were incubated at room temperature in the dark for 5 min in a 2% Alizarin Red S (pH 4.25) solution (Sigma-Aldrich; cat. no.: A5533). Finally, after sequential large-volume washes with PBS, brightfield microscopy pictures were acquired for the visualization of calcium deposits. Gene-edited cell sorting, clonal expansion and screening The sorting of EGFP-positive HeLa cells and of mCherry-positive human myoblasts was performed through a BD FACSAria III flow cytometer (BD Biosciences) or a CytoFLEX SRT Cell Sorter (Beckman), after subculturing the transduced cells for >25 days. The individually sorted HeLa cells were plated in wells of 96-well plates containing a 1:1 mixture of regular culture medium and FBS with penicillin/streptomycin at 100 U ml-1. In addition, to increase the cloning efficiency, this mixture was supplemented with 50 μM α-thioglycerol and 20 nM bathocuprione disulphonate (both from Sigma-
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