Selection And Characteritation Of Effective Promoter In Bovine Myoblasts

There are more protein, lesser fat and cholesterol in beef and also lots of amino acids and microelements in it. Beef becomes the primal meats for people to consume. With the realization about nutrition, people are more and more paying attention to high grade beef. So breeding superordinary beef by transgenic technology becomes focal point of study. Considering of safety, the study of muscle effective and specific promoter becomes important. In this study, eukaryotic expression vectors were constructed by recombinant DNA technology, promoter was synthesised and enhancer was found by transcription factor-binding sites assay and sequence blast to construct a bovine muscle effective and specific promoter.5' regulation region of bovine skeletal alpha-actin, Mylpf and Ckm were cloned to pGL3-Basic vector respectively. Liposome technique was used to transfect Luxi cattle fibroblasts and myoblasts and Dual-Luciferase(?) Reporter Assay System was used to compare the activity of the 5' regulation region of alpha-actin, Mylpf and Ckm. In addition, 82bp and 148bp fragments of bovine skeletal alpha-action promoter were used as core promoter, and the core promoter and synthetic regulation fragment were used to construct synthetic promoter respectively. According to location of enhancer in mouse muscle creatine kinase, a gene fragment in bovine muscle creatine kinase was found by transcription factor-binding sites assay, in order to detect the activity of the fragment, the fragment was cloned to the upstream of the synthetic promoter. Liposome technique was used to transfect Luxi cattle fibroblasts and myoblasts and Dual-Luciferase(?) Reporter Assay System was used to assay the activities of the synthetic promoter and enhancer.Activities of 5' regulation region in alpha-actin, Mylpf and Ckm were compared, after transfecting into Luxi cattle myoblasts, the activity of alpha-actin promoter was highest. Muscle specificities of the three promoters were valued, after transfecting into Luxi cattle fibroblasts, the activities of the three promoters were analogous to pGL3-Basic, it suggested that the three promoters all showed muscle specificities. After transfecting into Luxi cattle myoblasts, the 82bp and 148bp core promoter had activity, and the synthetic regulation element could increase the activity of the core promoter 82 bp and 148 bp fragments by 140–400-fold and 2–3-fold respectively. The activity of the synthetic promoter used 82bp as core promoter was about 4–6-fold of alpha-actin promoter, while the activity of the synthetic promoter used 148bp as core promoter was lower than alpha-actin promoter. The enhancer found in bovine muscle creatine kinase could increase the activity of synthetic promoters by 1–3-fold. The activities of plasmids containing enhancer and synthetic promoter were about respectively 9–11-fold and 2–3-fold of alpha-actin promoter, and the synthetic promoter used 82bp as core promoter with enhancer exhibited expression levels was about one sixth of the CMV promoter. After transfecting into Luxi cattle fibroblasts, the activities of plasmids containing enhancer and synthetic promoter were both analogous to those in myoblasts, suggesting that the synthetic promoter and enhancer had none muscle specificity.In this study, the natural muscle promoters were compared. An effective synthetic promoter and an enhancer found in bovine muscle creatine kinase were obtained. The synthetic promoter and enhancer gained in the study provided important materials for expression of exogenous gene in bovine muscle and production of transgenic bovine.