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Cloning of Bovine Myf6 Gene and Expression of the Gene in Myoblasts

Author: TangZhanYi
Tutor: GaoXueJun
School: Northeast Agricultural University
Course: Biochemistry and Molecular Biology
Keywords: Cattle Myf6 Eukaryotic expression vector Myoblast Transfection Cell differentiation
CLC: Q78
Type: Master's thesis
Year: 2010
Downloads: 72
Quote: 0
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Abstract


With the changing of people's dietary structure and growing concern about the health of the beef quality has been unprecedented attention, high-quality, high-grade beef short supply. The myogenic family controls the entire process of muscle development, the styling of the front body muscle cells, proliferation and the formation of muscle fibers, myogenic regulatory factor involvement until maturity and improve the function of the individual after birth, there is a direct impact on animal production meat capacity and muscle quality. Myf6 myogenic family of a gene regulatory mechanism of muscle development, achieve of myf6 gene regulation through in-depth research myf6, you can change the traits of the muscle fibers in the process of muscle formation, thereby improving the quality of beef. The experimental application of PCR method the amplification myf6 gene fragment in the multiple cloning site of the plasmid pIRES2-EGFP construct the eukaryotic expression vector pIRES2-EGFP-myf6 insert myf6 gene. The liposome technology transfection Luxi cattle fibroblast cells and myoblasts stably transfected cell lines were screened by G418. Using western blot, Real-time PCR to detect cells before and after transfection myf6 gene, muscle creatine kinase gene and myosin light chain gene expression levels change. In this study, the initial establishment liposome method the transfection myf6 transgenic technology, the G418 fibroblasts minimum lethal concentration of 400μg/ml, the myoblasts minimum lethal concentration of 600μg/ml, two cell screening were obtained a stable transfected cell lines. Increases myf6 protein and mRNA expression in fibroblasts and myoblasts after transfection (P lt; 0.01), elevated muscle creatine kinase gene mRNA expression (P lt; 0.01), myosin light chain gene The expression level of mRNA also increased (P lt; 0.01). Cell morphology observation fibroblasts after transfection morphological changes unfused myotubes. Myoblast fusion to myotubes after transfection. Show that the eukaryotic expression vector pIRES2-EGFP-myf6 highly expressed in fibroblasts and myoblasts and myf6 gene promotes myoblasts to the differentiation of muscle cells. This study aimed to construct the eukaryotic expression vector by recombinant technology, the myf6 gene expressed in of Luxi cattle fibroblasts and myoblasts, to establish the technical basis of the myf6 gene transfected cells. The obtained gene myf6 data of the two cell differentiation. The cell lines obtained in this study the stably transfected myf6 gene provides important experimental material for the next turn myf6 gene beef cattle somatic cell cloning.

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