The Construction Of Muscle-specific Promoter IGF2 Expression Vector And Its Impact On Bovine Skeletal Muscle Satellite Cells Proliferation

IGF2(insulin-like growth factor 2) is a protein composed of 67 amino acid residues, it is an important regulator of skeletal muscle growth and development, it plays an important role in regulating cell proliferation, differentiation, and programmed cell death, individual growth and development. In recent years, the research on IGF2 is gradually deepening, however, by way of overexpression IGF2 to cultivate high-quality beef which is high-yielding and fast growth is less reported. This study aims to select IGF2 expression vector with efficient and specific promoter and study its effects on bovine skeletal muscle satellite cells proliferation. To improve the efficiency of IGF2 expression in muscle tissues, obtain a highly expressed vector containing IGF2 is essential, in which choice of the promoter is an important factor. Currently, cytomegalovirus(CMV) promoter is one of the promoters which are often used in foreign gene expressing in eukaryotic cells. CMV promoter, although efficient but almost no cell-specific, and taking into safety of genetically modified organisms, it can not be applied to the production of transgenic cattle. Therefore, the selection of efficient and specific promoter is necessary for the expression of foreign genes in eukaryotic cells. In this study, we use the three pre-built promoters which have a relatively high activity and specificity in laboratory: Desminpro(300) is a bovine desmin gene promoter in size 300 bp, its activity is higher, its ability to promote gene expression is twice as α-actin promoter and the creatine kinase promoter of human skeletal muscle; CMV-Myo Gpro is connected to some CMV enhancer sequence before Myo G gene promoter, the transcriptional activity of CMV-Myo Gpro complex promoter increased about 9 times as bovine Myo G promoter reaching a strong promoter CMV promoter 72% in C2C12 cells, compared with bovine skeletal muscle satellite cells, CMV enhancer enhance transcriptional activity is more effective; Myo Gpro-double is Myo G promoter fragment containing two copies of regulatory elements, its transcriptional activity was significantly higher than that of ordinary Myo G gene promoter, about 3.8 times as the Myo G gene promoter, the three promoters have a higher activity and better muscle-specific. In this study we obtained IGF2 eukaryotic expression vector containing the above three types of muscle-specific promoter, through transfection and expression of IGF2 testing, we screened IGF2 expression vector with a higher specific activity and stronger muscles specific, and explored its role in the regulation of bovine skeletal muscle satellite cells. The main findings are as follows;(1)The construction of muscle-specific IGF2 expression vectorThe experiment was according to the gene sequence of bovine IGF2 that Gene Bank published, primers were designed and bovine skeletal muscle satellite cell genomic DNA as template,bovine IGF2 gene was amplified by PCR, and constructed p GL3-desminpro-IGF2, p GL3-CMV-Myo Gpro-IGF2, p GL3-Myo Gpro-Double-IGF2 with three kinds of muscle-specific promoter IGF2 eukaryotic expression vector using three kinds of muscle-specific promoter :Desminpro, CMV-Myo Gpro, Myo Gpro-Double which are pre-builted in laboratory;(2)Screening the activity and specificity of the expression vectorInstantaneous transfected into bovine skeletal muscle satellite cells and bovine fetal fibroblast cells, the relative expression of IGF2 was detected by RT-PCR, the results showed that the expression levels of IGF2 in p GL3-desminpro-IGF2 group was significantly higher than the other two groups, and has better muscle-specific;(3)Ed U detect effects of p GL3-desminpro-IGF2 on cell proliferationAfter transfected p GL3-desminpro-IGF2 vectors into cells, EDU testing proved p GL3-desminpro-IGF2 can promote cell proliferation, compared with the control, the proliferation rate is 27.31%;(4)Related gene of expression changes were detected after p GL3-desminpro-IGF2 transfected into cellsThen we transfected PGL3-desminpro-IGF2 into bovine skeletal muscle satellite cells, the change of expression of cell cycle-related genes were detected by RT-PCR. The results showed that IGF2 promoted cell proliferation by downregulating the expression of P27 which is the inhibitor of CDKs, increasing the expression of CDK6. The results show that the gene accelerating cell proliferation may be by regulating AKT phosphorylation.The study obtained a high efficient and specific eukaryotic expression vector p GL3-desminpro-IGF2 which can highly efficient express in in bovine skeletal muscle satellite cells, it can significantly improve muscle cell proliferation rate. It provides important assistance to improve production of transgenic beef muscle.