The Effects Of RNAi On Expression Of Myostatin In Porcine Cells

RNA interference (RNAi) refers to the selective degradation of mRNA induced by double-stranded RNA (dsRNA), first discovered in Caenorhabditis elegans. Homology-dependent silencing phenomena related to RNAi have been observed in many species from all eukaryotic kingdoms. The hallmark of these phenomena is the presence of short dsRNA molecules (21–23 bp long), termed short interfering RNA (siRNA), which are generated from dsRNA by the activity of Dicer, a specific type RNAseIII. These molecules serve as a template for the recognition and cleavage of the cognate mRNA. RNAi approach provides a powerful method to study the functions of genes in mammals.Objective: To construct the expression short hairpin RNA (shRNA) targeting gene myostatin in the fetal fibroblasts and the myogenic Satellite cells of porcine, using psiSTRIKE, and to observe the effects on the expression of messenger RNA(mRNA) of gene myostatin. Methods: Three RNA interference (RNAi) sites targeting the myostatin gene was selected. Four pairs of oligonucleotides fragments were designed and synthesized,then them were annealed to double-strand, finally cloned them into the vectors containing U6 promoter,obtained the vector expressing four aim genes. Every experiment was divided into three groups: Experimental group, negative plasmid control group and normal control group. Reverse transcription-polymerase chain reaction (RT-PCR) was carried out to detect the expression of the mRNA of gene myostatin having been knocked down.Results: The expression of mRNA of gene myostatin of experimental groups were markedly lower than those of other two control groups (P<0.05). There was no statistically significant between two control groups. The RNA interfering effect in fetal fibroblasts was markedly higher than that in the myogenic Satellite cells (P<0.05).Conclusion: The three recombined interfering plasmids could suppress the expression of the mRNA of myostatin gene in porcine.