Abstract:

BACKGROUND: The treatment for skeletal muscle atrophy is a difficult and hot point both in basic and clinical researches. In recent years, gene therapy has aroused extensive attention. 
OBJECTIVE: To construct a recombinant plasmid for expressing the rat skeletal α-actin/human insulin-like growth factor-1 (RSKA/hIGF-1) gene and to observe its expression in C2C12 cells.
METHODS: The construct consists of rat skeletal a-actin promoter sequence, hIGF-1 coding sequence and human growth hormone 3’UTR region. The construct was initially cloned into a pBluescript Ⅱ SK(+) after synthesized, which contains the plasmid origin of replication and Ampicillin resistance gene. The sequence of synthesized hIGF-1 was confirmed by restriction analysis and DNA sequencing, then transfected into C2C12 cell. HIGF-1 gene was detected by RT-PCR and hIGF-1 was found by Western blot.
RESULTS AND CONCLUSION: The restriction map of PBS-RSKA/hIGF-1 was identical to expectation, and sequencing verified that the inserted fragment was not changed. Specific bands could be seen in transfected C2C12 cells by RT-PCR and Western blot. Recombinant plasmid PBS-RSKA/hIGF-1 constructed successfully and it can be effectively expressed after being transfected into C2C12 cells in vitro.