Preliminary Research on Knockout of Myostatin Gene in Cultured Sheep Fibroblasts
|School||Liaoning Normal University|
|Keywords||Sheep Myostatin Knockout Displacement-type knockout vector Fibroblast|
The myostatin prime (of myostatin in, MSTN), also known as of GDF-8 (transforming growth factor), for the first time at the Johns Hopkins University School of Medicine in 1997 Mcpherron research team in the study of TGF-β (transforming growth factor superfamily) amplification using degenerate PCR method in the conserved region of the family can be found. Negative regulation of the gene as a skeletal muscle-specific growth and development factor, genetic mutations can produce functional inactivation sharp increase muscle \The this study knockout technology vitro construct a third outside of the Sheep Myostatin gene exon deletion knockout vector transfected fibroblasts into sheep, the backbone vector the Neo gene replacement Myostatin gene exon3 to reach intracellular Myostatin gene inactivation. The sheep body cells Myostatin gene knockout feasibility preliminary exploration and research; lay the foundation to create new materials sheep breeding, transgenic sheep. The main findings of the paper are as follows: (1) sheep Myostatin gene sequence (GenBank No.DQ530260), LA-PCR technology, successfully amplified Sheep Myostatin gene homologous to the long arm 4.9k, including all of exon 1, intron 1, exon2, and part of the promoter and most intron2; homologous to the short arm of 1.1K, including part exon3 and 3 'untranslated region sequences, both connected to the PloxpII positive and negative screening gene knock inter backbone vector was successfully constructed specifically Myostatin first three exon regions displacement type knock missing in addition to the carrier PloxpII-OVIS-of MSTN (13.5K), digestion and sequencing analysis proved that the carrier is constructed correctly. (2) establish tissue adherence sheep ear marginal fibroblast cell line, proliferation activity, lay the foundation for subsequent cell transfection experiments. (3) to establish a sheep Myostatin knockout vector transfection and screening methods: liposomes 2.5ul, knockout vector 2ug, higher transfection efficiency; After 300ug/mlG418 initial screening of 7-10 days, then by 200ug/mlG418 The plus 50nmol/lGANC to maintain the screening of about 20 days can be significant cell clones. (4) After about 30 days the sign screening, 64 cell clones were obtained by PCR after the occurrence of the positive cells of the homologous recombination cloning initially considered the 12th cell clones. In addition to technical study the Myostatin gene mutation can lead to surge in muscle mass and a \transgenic sheep meat is high to establish a technology platform, to provide new ideas for the sheep breeding and breed improvement.