RNAi-mediated knockdown of mouse melanocortin-4 receptor in vitro and in vivo, using an siRNA expression construct based on the mir-187 precursor

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  • Kato Minoru
    Research Unit/Neuroscience, Mitsubishi Tanabe Pharma Corporation, 1000 Kamoshida, Aoba-ku, Yokohama, Kanagawa 227-0033, Japan
  • Huang Yi-Ying
    Laboratory of Chronogenomics, Mitsubishi Kagaku Institute of Life Science, 11 Minamiooya, Machida, Tokyo 194-8511, Japan
  • Matsuo Mina
    Experimental Animal Laboratory, Mitsubishi Kagaku Institute of Life Science, 11 Minamiooya, Machida, Tokyo 194-8511, Japan Present address: Division of Animal Experimental Laboratory, Life Science Research Center, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan
  • Takashina Yoko
    Pharmacology Laboratory, Mitsubishi Tanabe Pharma Corporation, 1000 Kamoshida, Aoba-ku, Yokohama, Kanagawa 227-0033, Japan
  • Sasaki Kazuyo
    Pharmacology Laboratory, Mitsubishi Tanabe Pharma Corporation, 1000 Kamoshida, Aoba-ku, Yokohama, Kanagawa 227-0033, Japan
  • Horai Yasushi
    Safety Research Laboratory, Mitsubishi Tanabe Pharma Corporation, 1000 Kamoshida, Aoba-ku, Yokohama, Kanagawa 227-0033, Japan
  • Juni Aya
    Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
  • Kamijo Shin-Ichi
    Experimental Animal Laboratory, Mitsubishi Kagaku Institute of Life Science, 11 Minamiooya, Machida, Tokyo 194-8511, Japan Present address: Division of Planning of Laboratory Animal Facility, Lifewill Corporation, 2740-12 Shimo-oyamada, Machida, Tokyo 194-0202, Japan
  • Saigo Kaoru
    Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
  • Ui-Tei Kumiko
    Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
  • Tei Hajime
    Laboratory of Chronogenomics, Mitsubishi Kagaku Institute of Life Science, 11 Minamiooya, Machida, Tokyo 194-8511, Japan Present address: Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa, Ishikawa 920-1192, Japan

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タイトル別名
  • RNAi-mediated knockdown of mouse melanocortin-4 receptor <i>in vitro</i> and <i>in vivo</i>, using an siRNA expression construct based on the mir-187 precursor

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説明

<p>RNA interference (RNAi) is a powerful tool for the study of gene function in mammalian systems, including transgenic mice. Here, we report a gene knockdown system based on the human mir-187 precursor. We introduced small interfering RNA (siRNA) sequences against the mouse melanocortin-4 receptor (mMc4r) to alter the targeting of miR-187. The siRNA-expressing cassette was placed under the control of the cytomegalovirus (CMV) early enhancer/chicken β-actin promoter. In vitro, the construct efficiently knocked down the gene expression of a co-transfected mMc4r-expression vector in cultured mammalian cells. Using this construct, we generated a transgenic mouse line which exhibited partial but significant knockdown of mMc4r mRNA in various brain regions. Northern blot analysis detected transgenic expression of mMc4r siRNA in these regions. Furthermore, the transgenic mice fed a normal diet ate 9% more and were 30% heavier than wild-type sibs. They also developed hyperinsulinemia and fatty liver as do mMc4r knockout mice. We determined that this siRNA expression construct based on mir-187 is a practical and useful tool for gene functional studies in vitro as well as in vivo.</p>

収録刊行物

  • Experimental Animals

    Experimental Animals 66 (1), 41-50, 2017

    公益社団法人 日本実験動物学会

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