Dystrophin Gene Deletion in DMD Cells

HEK293T cells were obtained from the American Tissue Collection Center (ATCC) through the Duke Cell Culture Facility and were maintained in DMEM supplemented with 10% fetal bovine calf serum and 1% penicillin/streptomycin. Immortalized myoblasts^{70 (link)} from a DMD patient harboring a deletion of exons 48–50 (Δ48–50) in the dystrophin gene were maintained in skeletal muscle media (PromoCell) supplemented with 20% fetal bovine calf serum (Sigma), 50 μg/ml fetuin, 10 ng/ml human epidermal growth factor (Sigma), 1 ng/ml human basic fibroblast growth factor (Sigma), 10 μg/ml human insulin (Sigma), 1% GlutaMAX (Invitrogen), and 1% penicillin/streptomycin (Invitrogen). All cell lines were maintained at 37°C and 5% CO₂. HEK293T cells were transfected with Lipofectamine 2000 (Invitrogen) with 400 ng of each expression vector according to the manufacturer’s protocol in 24 well plates. Immortalized myoblasts were transfected with 5 micrograms of each expression vector by electroporation using the Gene Pulser XCell (BioRad) with PBS as an electroporation buffer using optimized conditions ^{28 (link)}. Transfection efficiencies were measured by delivering an eGFP expression plasmid (pmaxGFP, Clontech) and using flow cytometry. These efficiencies were routinely ≥ 95% for HEK293T and ≥ 70% for the immortalized myoblasts.