Building Of The Bovine CMYA1 Core Promoter Vector And Building Of The Mouse Early Embryos

CMYA1 is an actin-binding protein that plays a vital role in cardiac morphogenesis. CMYA1 is expressed specifically in the myocardial and skeletal muscle and is up-regulated in injured muscle. In order to get effective vector which can change bovine muscle specific traits, this experiment through the core of the CMYA1 gene promoter connecting the gene encoding diacylglycerol acyltransferase(DGAT1) to build the vector. Establish the foundation for obtain high yield and good quality transgenic cattle in the futureIn the first place, identification the CMYA1 gene core promoter specificity in the bovine muscle. p EGFP vector which builded by our laboratory transfected into the bovine myoblast cells and bovine fibroblasts cells. The result proved that EGFP only expressed in the bovine myoblast cells. To evaluate the activity of the CMYA1 core promoter, the CMYA1 core promoter and DGAT1 gene were cloned into a pc DNA3.1 vector, which named XD. The muscle creatine kinase(MCK) promoters and DGAT1 gene were cloned into a pc DNA3.1 vector in the before, which named A293-4. XD A293-4 pc DNA3.1 vectors were transfected into C2C12 cells. The expression levels were measured using real-time PCR. The CMYA1 promoter drove the expression of the target gene six times higher than did the MCK promoter and 30 times higher than did the pc DNA3.1. Verify the influence of XD vector to the development of mouse fertilized eggs by pronuclear microinjection. The result show that the best injective concentration is 2 mg/l and the best site is male pronucleus. The XD vector can be useful in the building of transgenic mouse model in the near future in our laboratory.Conclusion: 1 The bovine CMYA1 core promoter was expression-specific in bovine myoblast cells. 2 The promoter of bovine CMYA1 would be an excellent effective promoter which can drive the DGAT1 gene expression in bovine myoblast cells. 3 Successfully got the early mouse embryos.The significance of the study is that obtained the effective vector which can change the bovine muscle traits. The experiment noly only build the foundation for the next step to obtain the transgenic bovine which have the good meat, but also provide the tool which have high efficiency and specificity to the researcher.