2.5 Plasmid construction and transfection
The PCR amplicons were ligated into eukaryote expression vector pcMINI using restriction enzymes Kpn I and EcoR I to construct the recombined pcMINI-DMD-wt/mut plasmids, which were transformed into E. colicompetent cell DH5α individually. The plasmid containing E. colicolonies were screened and the presence of inserted DMD gene fragments were determined by colony PCR using primer sets of pcMINI-DMD-KpnI-F and pcMINI-DMD-BamHI-R. The positive colonies were used for extracting plasmid by Rapid Mini Plasmid Kit (Simgen, Hangzhou, China) and plasmid was subsequently sequenced to confirm the inserts. The confirmed plasmids were followingly introduced into human embryonic kidney 293T cells and MCF-7 cells. The 293T cell line was cultured in DMEM medium containing 10% FBS (fetal bovine serum) and 1% penicillin/streptomycin at 37°C in the 5 % CO2 atmosphere. The MCF-7 cell line was cultured in MEM medium containing 10% FBS, 0.01mg/mL insulin, and 1% penicillin/streptomycin at 37°C in the 5 % CO2atmosphere. Liposomal Transfection Reagent (Yeasen, Shanghai, China) was used to transfect cell lines with pcMINI- DMD -wt/mut or pcMINI-C- DMD -wt/mut. After 6 hours of transfection, the transfect cells were re-suspended in fresh media and cultured for 48 hours. Total RNA was extracted from the enriched transfect cell lines and used for analysis later.