Construction And Validation Of Skeletal Muscle-Specific Reporter Gene Retroviral Vector

Objectives: Skeletal muscle satellite cells are considered as proliferating and differentiating myogenics cells in adult skeletal muscle cells. Establishing the simple and fast method to identify and filter skeletal muscle satellite cells has the practical meaning for culture and research and application of skeletal muscle satellite cells. As so far, we know that the expression of desmin is initiated in skeletal muscle satellite cells and it expresses to high level as a symbol protein in muscle myoblast fiber. So, regulating reporter gene by desmin's promoter is likely to establish simple methods to identify and filter skeletal muscle satellite cells. A reporter gene vector with desmin's promoter and enhancer was constructed and validated whether it expressed specially in skeletal muscle satellite cell or not by checking green-fluorescent proteins (GFP) 's expression. At the base of its right construction, retroviral supernatants were prepared and checked its expression level in differentiated P19 cells.Methods: (1) The construction and identification of recombined vector: First, the required DNA segments, GFP and neomycin gene (GFP+NEO) and the promoter and enhancer of desmin (pro+enh), were got by PCR, then restriction endonuclease and T4 ligase were used to make the required gene connect to linear segment of retroviral psuper, which was cut by Nco I, BamH I ,EcoR I and Sal I . (2 ) Validation of the recombined vector's expression speciality: Recombined vector and psuper were transfected into CHO and C2C12 cells, after 24 h, GFP was observed by converted fluorescence microscope and the pictures were captured.(3) Retroviral supernatants were prepared and transfected into C2C12 and P19 cells. After 24h, GFP were observed by converted fluorescence microscope and the pictures were captured.Results: (1) Every step of recombined vector's construction was identified by telectrophoresis, and it confirmed that recombined vector was constructed correctly.(2) After being transfected into CHO and C2C12 cells, recombined vector and psuper expressed GFP at the same level in C2C12 cells. But recombined vector didn't express in CHO and psuper expressed at a high level. (3) Retroviral supernatants was prepared and being transfected into C2C12 cells and P19 cells, the expression of GFP+NEO in C2C12 is a little higher than that of plasmid, but not more. GFP was observed in differented p19 cells, which indacated that DMSO could induce p19 cells to different into skeletal muscle cells. Conclusions: The telectrophoresis analysis identified that recombined vector was correct. The result of transfection showed that recombined vector had the expression speciality in skeletal muscle cells and retroviral supernatant was prepared, which were the base of the purification of skeletal muscle satellite cell and gave the new hope to tissue engineering and gene therapy.