Preliminary Research On Knockout Of Myostatin Gene In Cultured Sheep Fibroblasts

Myostatin (MSTN) also known as GDF-8 (growth and differentiation factor-8) which was first to be identified in using degenerate PCR technique when amplified the conserved regions of TGF-β(transforming growth factor-β) family by Mcpherron (Johns Hopkins University School of Medicine research group) in 1997. Research showed that fuctional inactivation of genetic mutations of myostatin could lead to muscle quantity surge to produce"double muscled"phenotype characteristics and as a specific negative regulators of skeletal-muscle development. Gene targeting was utilized to construct the knockout vector for devitalization myostatin exonⅢin vitro for increasing the meatyield. That laid the foundation for obtaining the transgenic sheep and creating new materials of mutton sheep breeding. The feasibility of myostain gene deletion in sheep somatic cell was preliminary explored. The results of the research as follows:(1) According to the sheep myostatin entire sequence (GenBank accession No.DQ530260), sheep myostatin gene homologous arm sequence was successfully amplified in using LA-PCR with optimized molecular cloning technology, the 4.9kb homologous long arm contained the whole exon1, intron1, exon2, parts of the promoter and most of the intron2 while the 1.1kb homologous short arm contained the partial exon3 and 3'UTR sequence. The myostatin gene was replaced by neomycin phosphotransferase gene after the homologous arm cloned into PloxpII vector, for inactivating their function and achieving the knock-out purpose. PloxpII-OVIS-MSTN(13.5K),which was replacement type knock-out vector specifically to deletion of exon3 of myostatin gene proved to be correct by restricted endonucleases digestion and sequence analysis.(2) Texel ear marginal tissue fibroblast cell line was successfully established by using a primary explant technique. That laid foundation for cell transfection experiment for their well proliferation activity.(3) Sheep myostatin gene targeting transfection and drug selecting method with higher transfection efficiency were established after optimizing the transfection condition: lipfectamine/targeting vector: 2.5ul/2ug.Cell clones were obviously emerged after initial selecting with 300ug/ml G418 for 7-10 days and then preserved selecting with 200ug/ml G418, 50nmol/l GANC for about 20 days. (4) 64 clones were obtained after the positive and negative selection for 30 days and No.12 cell clone was initially identified as homologous recombination positive clone after PCR amplification.Sheep myostatin gene replacement type knock-out vector was constructed, according to myostatin mutations that lead to muscle quantity surge to produce"double muscled"phenotype characteristics. This research had built a technical research platform for making transgenic sheep with higher meat production rates by gene targeting and nuclear transfer which providing a new idea for sheep breeding and breeding improvement. .