Inhibition Of Myostatin (MSTN) Expresstion In Primary Porcine Fetal Fibroblasts By Lentivector-mediated RNAi

Myostatin (MSTN) belongs to the transforming growth factor (TGF)-βsuperfamily, is a well-established negative regulator of skeletal muscle mass. Dysfunction of MSTN gene either by natural mutation or induced through genetic manipulation (knock out or knock down) has been reported to increase the muscle mass in mammalian species. RNA interference (RNAi) is a sequence-specific posttranscriptional gene silencing mechanism initiated by the introduction of double-stranded RNA (dsRNA) into target cells. Delivery of siRNAs into mammalian cells by transfection with DNA plasmids expressing short hairpin RNAs (shRNAs) has been shown to mediate RNAi successfully. Importantly, recent reports describe the use of viral vectors to deliver siRNAs successfully into mammalian cells in order to get stable gene silencing. The expression of shRNAs with lentiviral vectors is useful to induce stable RNA interference. Myostatin shRNA gene transfer can be used as a potential strategy to increase muscle mass. Thus, we decided to investigate the feasibility of applying lentivectors expressing MSTN-specific shRNA in primary porcine fetal fibroblasts for silencing of the myostatin gene. The objective of the present study was to knock down myostatin gene expression in primary PEFs, in order to provide a basis for studying further details of the role of MSTN in porcine muscle development. Using this approach, we examined the effect of myostatin knockdown on primary PEFs. We report that up to 97% silencing of myostatin mRNA was achieved using shRNA constructs in transiently fetal fibroblasts (p<0.05). For lentivector-mediated RNAi, Real-time PCR revealed that cells stably transduced with lentivectors resulted in an efficient and stable suppression of myostatin accumulation, up to 94% of the myostatin mRNA was silenced (p<0.05), compared with the non-transduced controls. Adverse effects of RNAi were investigated in this study. We demonstrate that none of the two shRNA constructs we used in this study induced high level of IFN-βor OAS2 expression in transiently transfected fetal fibroblasts. However, contrary to our expectations, highest IFN-βand OAS2 level were obtained in stably transduced cells (2.49-fold and 14.43-fold increase respectively, p<0.05), as to non-transduced controls. In conclusion, myostatin gene knockdown was effectively performed by lentivector-mediated delivery of shRNA in this study, but reduction of the interferon response will be essential for long term applications. Furthermore, the obtained shRNA-expressing lentiviral vectors can be used for producing transgenic embryo, in order to produce double muscling animals that can transmit to future progeny.