Study On The Method Of Editing Chicken MSTN Gene And MF2C Gene By CRISPR/Cas 9

Myostatin(MSTN)is a negative regulator of muscle growth.It is also an important regulator gene that affects lean meat rate and muscle quality of livestock and poultry in recent years.Mef2 C gene belongs to Mef2 family.Mef2 C gene is involved in the formation of skeletal muscle fibers and promotes the formation of slow muscle fibers.In order to obtain genetically modified chickens with delicious meat and stable genetic expression,we used CRISPR/Cas 9 gene editing technology to construct MSTN gene Cas 9 knockout vector and Mef2 C gene g RNA-Cas 9 inserted into eukaryotic expression vector and Mef2 C gene targeting vector.The method of CRISPR/Cas 9 editing chickens was studied from cell level to individual animal.Firstly,chicken primary myoblasts were isolated and transfected on the basis of Cas 9 knockout vector of MSTN gene and g RNA-Cas 9 of Mef2 C gene inserted into eukaryotic expression vector and Mef2 C gene targeting vector.Quantitative analysis of myogenic regulatory factors was carried out after transfection to observe the proliferation of Ed U and CCK8 cells.The results showed that Cas 9 knockout vector of MSTN gene down-regulated the expression of MSTN gene,while myogenic regulatory factors My F5 and Myo D were up-regulated.The results of proliferation test of Ed U and CK8 cells showed that knockout of MSTN gene promoted myoblast proliferation;g RNA-Cas 9 of Mef2 C gene was inserted into eukaryotic expression vector and Mef2 C base.Mef2 C gene expression was significantly up-regulated by targeting vector,myogenic regulators My F5,Myo D and My HC were up-regulated.The results of proliferation test of Ed U and CCK8 cells showed that insertion of Mef2 C gene promoted myoblast proliferation.Mef2 C gene g RNA-Cas 9 inserted into eukaryotic expression vector and Mef2 C gene targeting vector can promote cell proliferation better than MSTN gene Cas 9 knockout vector.Chicken sperm cells were cultured and transfected in semen dilution solution,and the viability of sperm cells was counted for 1-6 hours.After 6 hours of transfection,green fluorescence was found in chicken sperm cells and some sperm cells were still viable after 6 hours.In the comparison of liposome transfection and electrotransfection of DF-1 cells,it was found that the effect of electrotransfection was better than that of liposome transfection.According to Neon Transfection System,the optimum conditions of sperm cell transfection were explored,and animal experiments were carried out.Liposome sperm vector method,electro-transfection sperm method and microinjection lentivirus method were used to prepare transgenic chickens,but CRISPR/Cas 9 edited chickens were not obtained.