The Establishment Of Myostatin Knocking-out Swine Fibroblast Cell Lines And They Developed In Vitro

Myostatin is one member of transforming growth factors beta(TGF-β) family which is also called growth or differentiation factor–8(GDF-8) family. It plays an essential role in regulating skeletal muscle growth. In nature, the Belgian blue and Piedmontese have double muscle phenotype, as the mutation of Myostatin.The discovery of Myostatin provids new scientific research route for breeding worker and vigorous fact base for further boosting animal performance of producing meat, and infarctate rationale for directive breeding on molecular level. so Breeding workers beside of carrying out propagate utilize forthcoming double-muscled animal, They are also knocking-out Myostatin by gene targeting, to get transgenic animal, in order to produce livestock which have more muscle and delicious.Now, Gene targeting is becoming one of thet method to study the function of genes. It is a kind of new technology that developed after 1980'based on homologous recombination.Using this technology we can alterspecific endogenous genes that mean we can change the genetic information of the animals at will. Through specific mutating the gene of the animals, we can know more about the function of the geneTaking the tissue of 35 days Yorkshire as materials, fibroblast lines were established by trypsin digestion and tissue attached culture method respectively In the study, targeting vector was introduced into fetal fibroblast of Yorkshire by lipofectamin-mediated DNA transfection methods, transfection parameters were optimized. Lipofectamin , plasmid vector , time of stable transfection, concentration of selected and keeped by G418 are optimized condition for transfection. At last,the positive cells were identified by PCR. Deceived of the positive cells were used for nuclear transfer.The donor cells were inserted into enucleated to reconstruct embryos. They developed in vitro.Fibroblast lines were successfully established by trypsin digestion and tissue attached culture method respectively In the study.Lipofectamin 4μl, plasmid vector 2μg, stable transfection 6 hours are optimized condition for transfection. After transfection, selecting with G418 (300μg/ml) for 7 days, then go on select with 200μg/ml G418+2μMGANC, the result is better. Deceived of myostatin knocking-out swine fibroblast cell lines. identification,the positive cells were used for nuclear transfer. reconstructed embryos developed blastula in vitro.