Suppression Of Myostatin Gene Expression With Vector-based RNA Interference Technique

Myostatin is a member of the transforming growth factor-beta (TGF-beta) family that functions as a negative regulator of skeletal muscle development and growth.RNA silencing or interference(RNAi )is a remarkable type of gene regulation based on sequence-specific targeting and degradation of RNA.The RNAi encompasses related pathways founding abroad range of eukaryotic organisms,including RNA interference of animal, quelling of fungi, posttranscriptional gene sileneing and cosuppression of genes in plants,etc. It plays important role in immunity against exogenous genetic factors (such as retrotransposons transgen and virus) and regulates gene expreosion of eukaryotic organisms. Silencing triggers are nuclcie acids which are converted into 21nt-long"small RNAs"in vivo. All RNA silencing pathways are triggered by"small RNAs"a term that encompasses small interfering RNAs (siRNAs).Objective: To construct the expression vector of siRNA targeting myostatin gene of ovis, and to observe the inhibitory effect of the vector on myostatin gene expression in C2C12 cells and in mice.Methods: 55 base-pair oligonucleotide for small interfering RNA expression targeting ovis MSTN gene was designed and chemically synthesized. After annealing, double oligonucleotides were inserted into the down-stream of CMV promotor of pSilencer4.1 to recombine pSilencer-mstn-siRNA plasmids. Eight siRNA expression vectors were transiently transfected into mouse fibroblast cell line C2C12 with lipofectamine-mediated transfection. RT-PCR and real time PCR was used to detect the expression levels of MSTN gene. Then the most efficacious siRNA used in vivo. Myostatin or a randomer negative control siRNA plasmid was injected and electroporated into the gastrocnemius muscle of mouse. The mice were sacrificed after 2 weeks. Myostatin expression was determined by real time PCR and immunohistochemistry;Muscle fiber size was determined by histochemistry.Results: The recombined psi-mstn-717 and psi-mstn-986 vector significantly suppressed the expression of MSTN mRNA by 61% and 53% in cells ,and gastrocnemius muscle fiber size increased by 33% and 44% to control after injection in vivo. Conclusion: The constructed siRNA expression vector of MSTN gene can block the expression of MSTN gene in vitro.