[Experimental study on micro-dystrophin gene transfection into C57/BL10 mice's myoblast]

To investigate the expression of micro-dystrophin gene in myoblast cultured in vitro, to explore the possibility of combining myoblast transplantation with gene transfer for Duchenne muscular dystrophy therapy.

Competent Escherichia coli JM109 was prepared, which transformed with plasmid pSL139, and positive clones were picked to cultivate. Plasmid was extracted with Alkaline lysis method and cutter with both Pvu I and Cla I enzyme. Agarose gel electrophoresis was employed to take pictures. Ten healthy 5-7 days old male C57/BL10 mice were selected, weighing 4-5 g, the primary and subcultured myoblasts were cultured with multi-step enzymatic digestion and differential adhesion method, and Desmin immunofluorescent method was used to identify. The 3rd generation myoblasts that were transfected with plasmid pSL139 mediated by liposome served as the experimental group, untransfected cells served as the control group. After 48 hours of transfection, the expressions of micro-dystrophin mRNA and protein in myoblasts were detected with RT-PCR and cell immunofluorescent methods, and the transfection efficiency was calculated.

After pSL139 plasmids being digested and for 40 minutes agarose gel of electrophoresis, 3.75 kb fragment of target gene and vector were observed. The cells were almost uniform, and triangular or diamond shape after 24-48 hours of culture; the cells turned to fusion manner and could be passaged after 4-6 days. Desmin immunofluorescent result showed that green fluorescence was seen in cytoplasm of most 2nd myoblasts, and the purity of the myoblasts was above 90%. At 48 hours after transfection of myoblasts with plasmid pSL139, RT-PCR results showed that about 300 bp fragment was seen in the experimental group and the control group, and the brightness was higher in experimental group. Immunofluorescent staining displayed that green fluorescence was seen in the cytoplasm of the myoblasts in the experimental group and no green fluorescence in the control group; the expression efficiency of positive cells for micro-dystrophin was 45%-55% in experimental group.

Micro-dystrophin gene can highly express at the levels of mRNA and protein respectively in myoblasts transfected with plasmid pSL139 mediated by liposome.