Chapter 4 —— 85 —— ATAG-3′, respectively. PCR products specific for EGFP served as internal controls for the availability and integrity of transgenic DNA and were amplified with primers #978 and #979 with sequences 5′- GAGCTGGACGGCGACGTAAACG-3′ and 5′-CGCTTCTCGTTGGGGTC TTTGCT-3′, respectively. RT-PCR analyses of gene-editing experiments Total RNA from myotubes differentiated from unmodified and DMD edited myoblasts was extracted with TRIzol Reagent (Thermo Fisher Scientific; cat. no.: 15596018) according to the manufacturer’s recommendations. Next, 500 ng of the resulting RNA was reversely transcribed into cDNA by using the SuperScript II Reverse Transcriptase kit (Thermo Fisher Scientific; cat. no.: 18064022) and Random Hexamer Primers (Thermo Fisher Scientific; cat. no.: SO142). Next, Platinum SuperFi II DNA Polymerase (Thermo Fisher Scientific; cat. no.: 12361010) was used for multiplexing PCR assays targeting edited and endogenous DMD transcripts. The compositions of the RT-PCR mixtures and thermocycling parameters are described in the Supplementary Tables S11 and S12, respectively. Quantification of AAV vector DNA cell entry HeLa were seeded at a density of 8 × 104 cells per well of 24-well plates. The next day, the cells were transduced with AAV-HRS1 at 0.5 or 2.0 TU cell-1 in the absence or in the presence of AdVP.C9KARA at 5, 10 or 20 GC cell-1. At 3-, 6-, 12- and 24-h post-transduction, the cells were thoroughly washed by four sequential washes with large volumes of PBS, after which total DNA was isolated by using the DNeasy Blood & Tissue Kit protocol (Qiagen; cat. no.: 69506). AAV vector DNA levels were determined by qPCR using iQ SYBR Green Supermix (Bio-Rad, cat. no. L010171C) together with the EGFP-specific primers #1243 (5′-CCATCCTGGTCGAGCTGG-3′) and #1246 (5′-CGGTGGTGCAGATGAACTTC-3′) at 0.3 μm each in a total volume of 15 μl. The PCR mixtures were subjected to an initial denaturation step at 95℃ for 5 min followed by 40 cycles at 95℃ for 10 s and 60℃ for
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