Construction Of Skeletal Muscle-specific Expression Vectors In Pigs

In order to improve the target gene expression leve in transgenic animals, it is necessary to construct some efficiency and specificityvectors.The selection of promoters is one of the important conditions.We add Enhancer and IRES to pDsRed-Express 2-1 vector to improve analysis of the promoters,which come fromαactin,CKM,CAPN 3,CA 3,TNNI 2,CASQ 1,GEM, The transcription factor binding sites of these promoters 5′flank are identified in silico,then we get some fragments with PCR and clone into the promoterless vector, Transient transfect vectors into myoblasts cells and other cells include Myocardial cells, kidney cells, endothelial cells, fibroblasts, liver cells,and induces myoblasts cells to myotubes.The results are following:1. The promoters ofαactin,CKM,CAPN 3 only are active in myotubes,these promoter are not active in other sources of cells.2. Other pig skeletal muscle-specificpromoters activity are not detected, nor in other sources of cells3. The promoter ofGEM isactivity in many kinds of cells.4. We get three pig skeletal muscle-specific expression vectors: CMV Enhancer-αactin Promoter-pIRES-DsRed, CMV Enhancer-CKM Promoter-pIRES-DsRed, CMV Enhancer-CAPN3 Promoter-pIRES-DsRed.5. We also get a non-specific expression vector: CMV Enhancer-GEM Promoter-pIRES-DsRedThese specific promoters are not active in many other tissues except for skeletal muscle. and could promoter the target gene expressed intransgenic animalsskeletal muscle.