Muscle Specific Bicistronic Genes MSTN SiRNA And HFAD3 Knockin At NCAPG-LCORL Locus In Cattle

Genome-editing technique can be used to achieve the multiple gene regulation and cultivating new varieties of genetically modified animals. The study was aimed to use the genome editing technique CRISPR/Cas9 to mediate muscle specific bicistronic gene(MSTN siRNA-hFAD3) knockin at NCAPG-LCORL locus which was identified as a safe transgenic locus in cattle, and to investigate the expression of muscle and fatty acids associated genes in the transgenic cells. In order to promot the muscle development and increasing the content of n3PUFA in muscle,effectively increase the amount of meat and improve the quality of meat. The results showed that (1) the muscle specific bicistronic gene MSTN siRNA-hFAD3 was successfully knocked in at NCAPG-LCORL locus. (2) The expressions of MSTN siRNA-hFAD3 in the transgenic cells were most likely muscle specific. (3) According to the results of the expressions of fatty acids related genes in the integrated bovine fetal fibroblasts and muscle satellite cells, the balance between fat synthesis and fat decomposition moved to the direction of adipose decompose, the expression of muscle development related genes were all raised, and the n6/n3 ratio was decreased. (4) Successfully obtain 6 strains of homologous recombinant monoclonal cell lines, and obtained the cloned embryos after transfer the transgenic cells into enucleated oocytes.In summary, the muscle specific bicistronic gene MSTN siRNA-hFAD3 expressed well in the transgenic cells, and it can be further used in production of transgenic cattle.