In vitro silencing of myostatin gene by shRNAs in chicken embryonic myoblast cells

Authors

  • Ajai K. Tripathi,

    1. Dept. of Animal Biotechnology, College of Veterinary Science & Animal Husbandry, Anand Agricultural University, Anand, Gujarat, India
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  • Mansi K. Aparnathi,

    1. Dept. of Animal Biotechnology, College of Veterinary Science & Animal Husbandry, Anand Agricultural University, Anand, Gujarat, India
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  • Amrutlal K. Patel,

    1. Dept. of Animal Biotechnology, College of Veterinary Science & Animal Husbandry, Anand Agricultural University, Anand, Gujarat, India
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  • Chaitanya G. Joshi

    Corresponding author
    1. Dept. of Animal Biotechnology, College of Veterinary Science & Animal Husbandry, Anand Agricultural University, Anand, Gujarat, India
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Abstract

RNA interference represents one of the potential mechanisms of regulation of gene expression. Selective downregulation of myostatin (MSTN), a member of transforming growth factor-β (TGF-β) superfamily and a negative regulator of myogenesis, has been demonstrated to enhance skeletal muscle growth. In this study, we studied short hairpin RNA (shRNA)-induced myostatin gene silencing in chicken embryonic myoblast cells using seven different shRNA-expressing constructs by reverse transcription-quantitative real time PCR (RT-qPCR). Myostatin-silencing efficiency of all shRNA constructs were first evaluated in human embryonic kidney cell line 293T (HEK293T) cells, where we observed 30–75.6% reduction in myostatin expression, followed by chicken embryo myoblast cells that revealed up to 55% reduction in myostatin expression along with upregulation of MyoD by 4.65-folds. Consistent with the earlier observations, the transfection of cells with plasmids led to significant increase in interferon responsive genes OAS1 and IFN β (2–112-folds), independent of myostatin silencing in both HEK293T and chicken embryonic myoblast cells. Our study suggests that apart from shRNA sequences, cell type-specific factors may play a significant role in determining the knockdown efficiency of shRNAs. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29: 425–431, 2013

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