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.