Determine The Effects Of MyoG And Myf6 Genes On Differentiation Of Bovine Skeletal Muscle Satellite Cells Using CRISPRi

Growth and development of skeletal mucsle relate to animal meat production and meat quality. As the adult muscle-derived stem cells, skeletal muscle satellite cells are widely used in in vitro studies of skeletal muscle development. The proliferation and differentiation of skeletal muscle satellite cells is a extremely complex biological process. The regulation of multiple transcription factors and cell signaling molecules is involved in these process. Muscle growth rely on the proliferation and differentiation of skeletal muscle satellite cells and further increase length and circumference of muscle fiber. MyoG(Myogenin), Myf6(myogenic factor 6), valued members of myogenic regulatory factor(myogenic regulatory factors,MRFs) family, play an important role in differentiation of muscle as regulator factors of skeletal muscle development. Recently, the CRISPR interference technique based on the development of CRISPR(clustered regularly interspaced short palindromic repeat) system become an important tool in studying gene function because it can interfere gene expression efficiently. MyoG and Myf6 are vital during differentiation of skeletal muscle satellite cells. In this study, MyoG, Myf6 genes were interfered by CRISPRi in bovine skeletal muscle satellite cells for exploring changes of muscle differentiation associated gene expression in differentiation process under the situation that MyoG, Myf6 genes expressed at low level. These would reveal MyoG, Myf6 genetic function and relationship in muscle differentiation process. We built MyoG,Myf6 gene interference vector useing CRISPRi to transfect bovine skeletal muscle satellite cells respectively,induced differentiation and selected vectors with the most eff icient interference differentiation through Real Time PCR and Western Blot,then use the vector to interfere MyoG alone; interfere Myf6 alone; interfere MyoG and Myf6 simultaneously, interfere MyoG and overexpress Myf6 simultaneously. We detect and add up the formation of myotube via immunofluorescence, and Real Time PCR and Western Blot were used in detecting expressing of important functional genes,namely MyoG, Myf6, MYH2 and Myo D in m RNA and protein level respectively.The results obtained are as follows:⑴ Once interference MyoG, Myf6 expression grow, Myf6 expression presented compensatory phenomenon,MYH2 expression decline, MyoD expression increse; and the more efficient, Myf6 is raising more, showing a clear compensation effect;⑵Once interference Myf6, MyoG and MYH2 expression decrease, Myo D expression rise;⑶Once interference both MyoG and Myf6,MYH2 expression drop,Myo D up-regulation;⑷Once interference Myf6, MyoG had no compensatory effects on Myf6, MyoG expression was decreased,the adaptation Myf6 and MyoG is one way non-reciprocal;⑸ Once interference MyoG, although Myf6 exhibit compensatory upregulation, the expression of muscle differentiation marker gene MYH2 reduced, indicating that Myf6 compensatory effects was not sufficient to fully compensate for MyoG on the effects of cell differentiation;⑹Once interference MyoG and Myf6 gene case severely hampered on the differentiated cells, significantly decreased the expression of MYH2, but compared with the separate disturbances,showed no additive effect;⑺When knockdown MyoG or Myf6, Myo D expression levels both increase;⑻The differentiation state of cells have not improved after knockdown MyoG and overexpressed Myf6 simultaneously, indicating that in case of low MyoG expression, overexpression of Myf6 cannot promote cell differentiation, MyoG play an irreplaceable role in the differentiation of cells.This study provide the theoretical basis for the study on the mechanism of muscle development, and providing reference for genetically engineered meat traits of poultry and increasing lean meat percentage of livestock production.