The Construction Of Expression Plasmid Containing Murine MSTN Full Coding Sequence And Its Identification In Vitro

Object:Obesity has emerged as a worldwide health issue, and has close relationship with many diseases, including type2diabetes, hypertension, coronary heart disease, stroke, dysregulation of lipid metabolism, insulin resistance, non-alcoholic liver disease, asthma and arthritis, etc. The much more fat accumulation in adipocytes is the core factor in occurrence of development of obesity. In1997, myostatin was found by American scientists, which could inhibit the growth of skeletal muscles. So far, the skeletal muscles were considered as one of the most important issues that consume the energy, and without the function of inhibiting muscles growth by myostatin may increase the energy consumption in muscles. The pathway probably indicates a new direction to study the pathogenesis of obesity. In this study, we constructed pcDNA3.1(+)-mMSTN vector containing murine MSTN full coding sequence. We will observe the influence of MSTN overexpression on the weight, epididymal fat, glucose and lipid metabolism in obesity mices in the future. Furthermore we will compare the relationship between MSTN level and obesity.Methods:1The construction of pcDNA3.1(+)-mMSTN plasmid containing murine MSTN full coding sequence:Total RNA from BALB/c mice were extracted from murine liver, transcribed reversly into cDNA, then amplified and gained high copies of murine MSTN cDNA full sequences. The pcDNA3.1(+) plasmid were performed to high copies. BamH I、 Apa I were used to cut the plasmid to have the vector fragments. Then we ligated the murine MSTN cDNA sequences and the vector fragments by T4lignase and constructed the pcDNA3.1(+)-mMSTN plasmid containing murine MSTN coding sequence successfully.2The identification of pcDNA3.1(+)-mMSTN plasmid containing murine MSTN coding sequence in vitro:The plasmid was transfected into murine3T3-L1cells by liposome transfection method and gathered the RNA from the cells after48hours, then observed MSTN expression of cDNA by real-time PCR method.Results:1Murine MSTN coding sequence was amplified successfully and ligated with vector fragments by T4ligase. The construction of pcDNA3.1(+)-mMSTN plasmid containing murine MSTN coding sequence was succeed.2pcDNA3.1(+)-mMSTN plasmid was transfected into murine3T3-LI cells by liposome transfection method. It was confirmed that pcDNA3.1(+)-mMSTN plasmid could well expressed in murine3T3-L1adipocytes..Conclusion:1mMSTN expression plasmid pcDNA3.1(+)-mMSTN was constructed successfully and could express murine MSTN protein in vitro murine preadipocytes3T3-L3cells and in vivo mices. This is a convenient tool for MSTN study.