Producing MSTN Gene Knockout Bama Pig By CRISPR/Cas9 Gene Editing

Bama mini pig is a characteristic local pig breed originated from Bama County,China.It has good flavor and strong resistance to adversity.However,it also shows low lean meat rate,high fat content,low feed conversion rate and slow growth rate in production.The traditional genetic improvement of pigs has a long period and high cost,and the gene editing technology represented by CRISPR / cas9 can efficiently and rapidly carry out targeted gene transformation of pigs,so it has great potential in the practice of pig genetic improvement.Myostatin is a known negative regulatory factor that affects the growth of animal muscles.Studies have shown that MSTN can significantly promote muscle growth and improve the lean meat rate of livestock,including pigs.The purpose of this study is to improve the growth speed and lean meat yield of Bama mini pig by editing MSTN with CRISPR / cas9.Firstly,the gene editing vector was designed,then MSTN gene editing renal fibroblast cell line was prepared by liposome transfection method,then the somatic cell nuclear transfer embryo was prepared with MSTN gene editing vector,and finally the gene editing Bama mini pig was produced by embryo transfer.The results showed that 7 cases of MSTN homozygous mutant cell lines were obtained after transfection with liposome.After the integration and miss target verification of two strains of cells,one was #1-5(?1/?1)and the other was # 1-10(+1/+1),1000 embryos were transferred to four surrogate sows,two of which were pregnant and finally gave birth to three cloned piglets,and two weaned females Bama pig were finally obtained the overall cloning efficiency of Bama mini pig was 0.3%.The living cloned pig was in good health,normal growth and development,and had significant difference in body weight compared with the wild type.There was no foreign gene integration and miss target phenomenon.Production tracking showed that the growth speed of MSTN knockout Bama mini pig was significantly faster,and showed obvious DMP.Further scale application of MSTN knockout pig will greatly improve the production efficiency of Bama mini pig and promote the development of related industries.