Construction Of A Gene Transfer Body Of MSTN RNAI Combined With Overexpression Of FSTN

Myostatin, also known as growth/differentiation factor-8(GDF-8) is a member of transforming growth factor β(TGF-β) family, mostly expressed in skeletal muscles and known to play a crucial role in the negative regulation of muscle growth. Follistatin (FSTN), also known as a suppressor protein of FSH, is a single chain glycoprotein. FSTN can combine with MSTN to block the function of MSTN, and promote muscle growth. MSTN knockout or knockdown and over-expression of FSTN can induce the target animals with significant increase in muscle masses. In this experiment, we constructed interference vectors of MSTN and detected the interference efficiency, and obtained two interference vectors which can disturb the expression of MSTN with high efficiency. Insert a bovine-derived MAR element into the interference vector with the highest efficiency to construct a MAR-containing interference vector. The presence of MAR element resulted in significant increased interference efficiency in both mRNA and protein levels when compared to the non-MAR containing vectors, which suggest that MAR enhance the interference efficiency. After infection of this gene transfer body to muscle satellite cells the genes associated with muscle development expressed increased in the MSTN knockdown cells. Finally, a FSTN gene was inserted to vector and constructed a gene transfer body pMAR-U6-shRNA926-CMV-FSTN, in which none bacteria sequence and resistant gene from the plasmid vector were existed. Using this gene transfer body we prepared and produced the transgenic mice which the transferred genes highly expressed in the founder mice. These results suggest that the bacteria sequence-free and resistant gene-free gene transfer body can be effectively expressed in transgenic mice. This study provides a safe and available protocol for construction of security and effective of knockdown and over-expressed vectors in animals. This gene transfer body can stable down regulate the expression of MSTN and up regulate the expression of FSTN.