Inhibition of myostatin gene expression in skeletal muscle of fish by in vivo electrically mediated dsRNA and shRNAi delivery.
Myostatin (MSTN), previously referred to as growth differentiation factor 8 (GDF8), is a negative regulator of skeletal muscle growth. In accordance with this role, natural mutations that inactivate the gene disrupting the function of the protein are associated with 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. If the atrophic actions of mammalian MSTN are indeed conserved in fish, strategies capable of inhibiting the expression of this gene could be applied to enhance growth performance in livestock production. Gene silencing by RNA interference has emerged as a promising new method of inhibiting the expression of targeted genes and inducing knockdown of associated proteins both in vitro and in vivo. Accordingly, we investigated here whether double-stranded RNA (dsRNA) or different plasmids expressing short-hairpin interfering RNAs (shRNAs) against myostatin and transduced by in vivo electroporation would increase skeletal muscle mass in reared European sea bass. After 7 weeks of intramuscular injections on a weekly basis followed by in vivo electrically mediated dsRNA delivery, no increase in the condition factor (K) of fish was observed as compared to the controls. Analogously, mean body weight and K of sea bass injected with three shRNAs were not higher than those of the control fish. On the other hand, MSTN transcript quantification via real-time RT-PCR revealed a significant inhibition of gene expression in the muscle of the dsRNA-injected fish and in the muscle of fish injected with one of the three tested shRNA-expressing vector constructs. In conclusion, in vivo electric-mediated delivery of dsRNA- or shRNA-expressing vectors against MSTN inhibits MSTN gene expression in adult sea bass muscle, but this is associated with an inconsistent double-muscle phenotype.
Department of Biotechnology and Life Sciences, University of Insubria, Varese, Italy. genciana.terova@uninsubria.it
Genciana
G
Terova
Simona
S
Rimoldi
Giovanni
G
Bernardini
Marco
M
Saroglia
D000818:Animals; D018274:Electroporation / Q000379:methods; D005399:Fishes / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D020868:Gene Silencing; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D018482:Muscle, Skeletal / Q000378:metabolism*; D055435:Myostatin / Q000235:genetics* / Q000378:metabolism; D010641:Phenotype; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics* / Q000378:metabolism; D014162:Transfection / Q000379:methods
D055435:Myostatin; D012330:RNA, Double-Stranded; D012333:RNA, Messenger; D034741:RNA, Small Interfering