Myostatin Gene Targeting In Cultured QinChuan Cattle Fetal Fibroblasts

Myostatin, a member of the transforming growth factorβ(TGF-β) family, is a negative regulator of muscle growth. The loss of this gene function has been associated with the phenotype described as"double muscling",an extreme form of muscle development characterized by a large increase in muscle mass. In our research, C313Y was implanted in QinChuan cattle myostatin, using gene targeting, so that it influenced the functions of Myostatin.Gene targeting was a technique for making transgenic animal that integrated exogenous gene into the expected site of cellular chromosome by homologous recombination to change animal's heritage characters. Gene Targeting had been usually used to reconstruct the gene in somatic cells. Two replacement vectors, pA2T-Mstn4.0 and pA2T-Mstn3.2, had been constructed in this paper. They had 4.0kb long arm and 3.2 long arm, respectively, and had the same 1.6kb short arm. The homologous sequences were generated by PCR from genome of Qinchuan cattle. A set of parameter(1250 V/cm;1ms;3 times) was optimized, used for electroporation. The two linearized vectors, pA2T-Mstn4.0 and pA2T-Mstn3.2, were transfected into the bovine fetal fibroblasts through electroporation. After selecting in cell culture medium with 600μg/mL G418 and 50 nmol/L GCV, we obtained 62 and 110 drug-resistant cell colonies, respectively. The NO.58 of them transfected with pA2T-Mstn4.0 was positive for targeted events after PCR identification, and the targeted colony was further confirmed by sequencing and Southern Blotting. This suggested that one allele of myostatin had been successfully mutagenized in bovine fetal fibroblasts.This research had built a technology platform for making transgenic beef cattle by gene targeting and nuclear transfer and accelerated to improve strain of beef cattle.