Construction Of Myod1Gene And Myog Gene Co-expression Vector And Effect Of Transfected On Fibroblasts Derived From Skin

Objective:It is a tough problem to treat denervated skeletal muscle atrophy. It is the best way for researching an approach of preventing of promoting muscle proliferation and differentiation to treat denervated skeletal muscle atrophy. People always treat denervated skeletal muscle atrophy by single gene. Now it has cooperation and facilitative effete between transcription factors through a long time study. Double genes can promote cell proliferation and differentiation, and can provide a new way of preventing denervated skeletal muscle atrophy.(1)To clone full-length rat Myodl and Myog cDNA, to construct recombinant cloning vector and to construct eukaryotic co-expression vector for further ex vivo gene transfection.(2) To establish an ideal cellular model of rat skin Fibroblasts for further ex vivo gene transfection.(3)To detect whether the eukaryotic co-expression vector of rat Myodl and Myog gene can properly express Myodl and Myog protein in the transfection experiment, and to investigate whether Myodl and Myog gene transfection can transform the primary cultured fibroblasts derived from rat skin into myoblasts.Methods:Full-length Myodl and Myog cDNA were amplified by RT-PCR. The PCR products were ligated into pMD18-T simple vector to generate the recombinant plasmid, designated pMD18-Myodl and pMD18-Myog, which was then by sequencing verified. The Myodl and Myog co-expression plasmid was generated by subcloning the Myodl and Myog cDNA into pVAXl. The recombinant Myodl and Myog were then transient transferred to primary culture fibroblasts derived from rat skin by transfection reagent. Expression of Myodl and Myog was detected by Western blot. Immunofluorescence was used to detect whether transfected cells were transformed into myoblast.Results:The sequencing results of pMD18-Myod1and pMD18-Myog and pVAXl-Myodl-IRES2-Myog-IRES2-EGFP were identical with reported rat Myodl and Myog cDNA sequence by Gene bank. The immunohistochemistry staining of vimentin and desmin confirmed that the primary cultured cells were fibroblasts and there was no myoblast contamination. Myodl and Myog expression was detected by Western blot after transient transfection. Immunofluorescence of desmin confirmed these cells were myoblasts. The pVAXl-Myodl-IRES2-Myog-IRES2-EGFP transfection could transform the primary cultured fibroblasts derived from rat skin into myoblasts.Conclusions:(1)The cDNA of Myodl and Myog, are cloned successfully.(2) The pMD18-Myodl and pMD18-Myog, cloning vectors for Myodl and Myog, are cloned successfully.(3)The pVAX1-Myodl-IRES2-Myog-IRES2-EGFP, a eukaryotic co-expression vector of Myodl gene and Myog gene, is successfully constructed.(4) The primary cultured cells from rat skin were fibroblasts only.(5) The pVAXl-Myodl-IRES2-Myog-IRES2-EGFP can be transfected into the primary cultured fibroblasts efficiently. The eukaryotic co-expression vector of rat Myodl and Myog gene can properly express Myodl and Myog protein in the transfection experiment.(6) Myodl and Myog gene transfection can transform the primary cultured fibroblasts derived from rat skin into myoblasts.