Gene silencing of myostatin in muscle of sea bass by in vivo electrically mediated dsrna and shrnai delivery.

Myostatin (MSTN), referred to as differentiation factor 8 (GDF8), is a secreted protein that acts as a negative regulator of skeletal muscle growth. Natural mutations of myostatin gene cause an excessive muscle growth and double-muscling phenotype in several mammalian species. Recent studies using transgenic MSTN deficient zebrafish and medaka support the idea that this gene inhibits skeletal muscle growth even in fish. These findings have suggested that strategies capable of inhibiting myostatin activity may be applied for enhancing growth performance in livestock production. RNA interference (RNAi) is one of the most promising and powerful method of inhibiting the expression of targeted genes both “in vitro” and “in vivo”. Accordingly, in this study we examined whether MSTN RNAi and intramuscular electroporation delivery, would locally increase sea bass muscle mass as a result of a decrease in the expression of MSTN gene. For silencing, dsRNA and different shRNA constructs (M1, M2, M3) were injected in the dorsal muscle of fish and then “in vivo” electroporation was applied for their delivery. After 7 weeks of intramuscular injections with dsRNA at a weekly basis, an increase in the condition factor (K) of fish was observed. Analogously, mean body weight and K of sea bass injected with one of the three shRNA-expressing vector constructs were higher than those of the control fish. MSTN mRNA quantification by real-time PCR revealed a significant inhibition of gene expression in the muscle of dsRNA injected fish (Fig. 1A) and in the muscle of fish injected with one of the three tested shRNA-expressing vector constructs (Fig. 1B). MSTN gene silencing was however not associated with excessive muscle growth and double-muscling phenotype in sea bass. In conclusion this study showed that in vivo electrically mediated delivery of dsRNA or shRNA plasmid vectors targeting MSTN, leads to the locally knock down of MSTN gene in sea bass muscle, but this reduction in transcript levels is not associated with excessive muscle growth. Nevertheless, due to the hypertrophic and hyperplasic postembryonic muscle growth, fish should provide an excellent experimental model for further elucidating the biochemical properties and signaling pathway of MSTN