Chapter 4 —— 79 —— T7 endonuclease I (T7EI)-based genotyping assays on amplicons spanning the target sites of the DMD-specific gRNAs. Lentiviral vector production and characterization The lentiviral vector transfer plasmids pLV.gRNADMD.IN3.1, pLV.gRNADMD.IN3.3 and pLV.gRNADMD.IN4.2 were assembled by inserting the gRNA expression units of, respectively, BC52_pgRNADMD.IN3.1, BC54_pgRNADMD.IN3.3 and BC56_pgRNADMD.IN4.2 (Supplementary information) into a lentiviral vector acceptor construct encoding the EGFP reporter. The corresponding vector particles were generated according to previously specified protocols [33]. In brief, 17 × 106 HEK293T cells were seeded in 175-cm2 culture flasks (Greiner Bio-One). The next day, 30 μg of a DNA cocktail diluted in 1 ml of NaCl and 90 μl of a 1 mg ml-1 PEI solution diluted in 1 ml of NaCl were mixed by dropwise addition of the PEI to the DNA under gentle agitation followed by vigorous homogenization with a vortex for 10 s. After an incubation for 15 min at room temperature, the DNAPEI complexes were directly added to the HEK293T cell culture medium. The DNA cocktail consisted of mixtures at 2:1:1 molar ratio of each lentiviral transfer plasmid, a packaging construct (psPAX2; Addgene #12260, a gift from Didier Trono) and a VSV-G-pseudotyping construct (pLP/VSVG; Invitrogen). At 2 days post-transfection, the conditioned medium was harvested, and the cellular debris was eliminated by centrifugation. The supernatants were then filtrated through 0.45-μm pore-sized cellulose acetate filters. Finally, the resulting clarified vector preparations were aliquoted and titrated by reporter-directed flow cytometry essentially as above-described for the titration of AAV vector batches. Viral vector transductions HeLa cells, hMSCs and human myoblasts were seeded at densities of, respectively, 5 × 104, 6 × 104 and 5 × 104 cells per well of 24-well plates (Greinder Bio-One) and, 16–18 h later, these cells were either mocktransduced or were transduced for 24 h with single or dual viral vector
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