Research Of The Interference Of The MicroRNA On Ovis Myostatin Gene

To clone sheep myostatin promoter for construction of short interference RNA(siRNA)expression vectors in sheep cells, a putative sheep myostatin promoter from sheep genomic DNA was amplified by PCR.After sequencing and bioinformatics analysis by online software revealed the presence of the critical transcription binding sites.The putative promoter was inserted into the reporter plasmid pEGFP-N1that can express green fluorescence protein and pEGFP-MSTN recombination vector was constructed. The transcriptional regulation activities of pEGFP-MSTN recombination vector were analyzed by detecting the fluorescence strength of EGFP in Sheep skeletal muscle satellite cells and skin flbroblast transfected with the vectors.The results indicated that the cloned pEGFP-MSTN recombination vector had the activity to switch on the EGFP expression in sheep skeletal muscle satellite cells while in Sheep skin flbroblast,transfection with pEGFP-MSTN recombination vector didn’t have the activity to switch on the EGFP expression. This suggested that the MSTN promoter had effective transcription activity and can be used for construction of short interference RNA(siRNA)expression vectors in sheep cells.To construct poMSTN-EGFP reporter vector for screening short interference RNA(siRNA)expression vectors in sheep cells, myostatin cDNA from sheep muscle was amplified by RT-PCR. After sequencing and the myostatin cDNA was inserted into the reporter plasmid pEGFP-N1that can express green fluorescence protein and poMSTN-EGFP recombination vector was constructed. The expression of poMSTN-EGFP recombination vector was analyzed by detecting the fluorescence strength of EGFP in mouse skin flbroblast transfected with the vectors. The results indicated that both the cloned poMSTN-EGFP recombination vector and pEGFP-N1had expression in mouse skin flbroblast. This suggested that the poMSTN-EGFP can be used for construction poMSTN-EGFP reporter vectorTo construct the expression vector of miRNA targeting myostatin gene of ovis and to observe the inhibitory effect of the vector on myostatin gene expression in mouce fibroblast cells. Five oligonucleotide for small interfering RNA expression targeting MSTN gene was designed and chemically synthesized. After annealing,double oligonucleotide were inserted into pcDNA5-miR to constrcut pcDNA5-miRNA-shRNA plasmids. Five miRNA expression vectors were transiently transfected into3T3cell line with lipofectamine-mediated transfection.The results showed that recombined pcDNA5-miR-shRNA197,pcDNA5-miR-shRNA364,pcDNA5-miR-shRNA369,pcDNA5-miR-shRNA380,pcDNA5-miR-shRNA484miRNA expression vector of MSTN gene can block the expression of MSTN gene in cell. Among pcDNA5-miR-shRNA364,pcDNA5-miR-shRNA369can siginificantly inhibit the expression of MSTN gene in cell according to fluorescence strength and quantity.