Objective: To construct a human source vector containing minidystrophin-EGFP fusion gene and investigate its expression in Cos-7 cells.
Methods: The recombinant human source vector named pHrnDysG was constructed with PCR-clone methods. Three fragments of dystrophin gene were PCR amplified from normal human dystrophin gene cDNA (GenBank NM04006). These three fragments were ligated to generate a minidystrophin gene. The enhanced green fluorescent protein (EGFP) gene was fused to the C terminal of the minidystrophin gene, and then the pHrnDysG was finally obtained by cloning the fusion gene to pHrneo. Fluorescence microscope and RT-PCR were used to detect the expression of minidystrophin-EGFP fusion gene after the recombinant construct was transfected into Cos-7 cells by lipofectamine.
Results: Restrictive enzyme digestion analysis and sequencing confirmed that pHrnDysG vector was constructed successfully. After the recombinant pHrnDysG was transfected to Cos-7 cells, RT-PCR demonstrated that the fusion gene was successfully transcribed, and the green fluorescence was observed at the cell membrane.
Conclusion: The minidystrophin-EGFP fusion gene mediated by pHrneo vector could express in Cos-7 cells and its products' localization in the cell membrane was the same as that of full length dystrophin. These results suggested that the recombinant human source vector pHrnDysG might be potentially used in studies on the gene therapy of Duchenne muscular dystrophy.