Effects Of Myomaker Gene Over-expression On The Expressions Of Some Membrance Fusion Related Genes

Myomaker is a muscle-specific membrane protein which is newly identified, and it is highly expressed during myoblasts fusion and is down-regulated after fusion. Over-expression of myomaker in myoblasts dramatically enhances the fusion effects of myoblasts. The research about myomaker is fewer and its action mechanism has not yet been elucidated till now. The objective of this study is to research the effects of over-expressiing myomaker on the mRNA expressions of the membrane fusion-related genes such as Dockl, Cdc42, Rac1, Trio, Myh9 and MyoF by constructing the myomaker expression vector then transfecting it into C2C12 cells. This study will lay a foundation for discover the molecular mechanisms of myomaker function.The first experiment, cloning of the CDS region of mouse myomaker:C2C12 cells were cultured and induced to differentiation, and then RNA were extractedand reverse transcribed into cDNA. The primers were designed according to the mouse myomaker gene registered in NCBI, then myomaker gene CDS sequence was amplified and was connected to the cloning vector pMD18-T Vector. The results of enzyme digestion and DNA sequencing revealed the construction is correct.The second experiment, construction of the myomaker expression vector:The myomaker primers added restriction sites were designed, thenthe myomaker gene CDS sequence was amplified from the pMD18T-Myomaker plasmid and connected to the expression vector pcDNA3.1 (+) for constructing the pcDNA3.1(+)-myomaker eukaryotic expression vector. The results of enzyme digestion and DNA sequencing revealed the construction is correct.The third experiment,influence of over-expressing myomaker in Dock1, Cdc42, Rac1, Trio, Myh9 and MyoF mRNA expression. The cultured C2C12 cells were divided into two groups by the different transfected plasmids, including the pcDNA3.1 negative control group and the pcDNA3.1-myomaker over-expression group. The transfected cells were variously treated after transfection as follows:some were cultured for 24h,48h or 72h with growth medium and others were cultured for 24h,48h or 72h with differentiation medium (2% horse serum). Then RNA were extracted from cells in six groups and the mRNA expressions of some membrane fusion-related genes were tested by the fluorogenic quantitative PCR. The results showed that:(1) Compared with the negative control group, the myomaker mRNA expression in the over-expression group had a significantly increase, which indicated we have successfully realized the myomaker over-expression.(2) Compared with the corresponding negative control groups, the changes of mRNA expression of some membrane fusion genes in myomaker-overexpressed groups are as follows:Cdc42 mRNA expression had significant decline in the differentiated cells at 24 hours and 72 hours; the mRNA expression of Racl had a significant increase in the undifferentiated cells at 72 hours but had significant decrease in differentiated cells at 24 hours and 72 hours; the mRNA expression of Myh9 had significant increase in the undifferentiated cells at 24 hours and in the differentiated cells at 72 hours; the mRNA expression of Trio had a significant decline at 24 hours in the undifferentiated and differentiated cells, but had significant elevation in undifferentiated cells at 48 hours and 72 hours; the mRNA expression of Dockl had a significant decrease in undifferentiated cells at 24 hours.