Lipofection of siRNA into bovine 8-16-cell stage embryos using zona removal and the well-of-the-well culture system

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  • IKEDA Shuntaro
    Laboratory of Animal Physiology and Functional Anatomy, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
  • SUGIMOTO Miki
    Laboratory of Animal Physiology and Functional Anatomy, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
  • KUME Shinichi
    Laboratory of Animal Physiology and Functional Anatomy, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan

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<p> Bovine preimplantation embryos exhibit dramatic biological changes between before and after the 8-16-cell stage. Here we report a simple lipofection method to transfect siRNA into bovine 8-16-cell stage embryos using zona removal and the well-of-the-well (WOW) culture system. Bovine one-cell embryos produced in vitro were freed from the zona pellucida and cultured up to the 8-16-cell stage in WOW dishes. The 8-16-cell embryos were lipofected with siRNA and the transfection efficiency was assessed at 48 h of transfection. Lipofection with a red fluorescent non-targeting siRNA revealed the importance of zona removal for transfection of siRNA into embryos. Using this method, we knocked down the methionine adenosyltransferase 2A (MAT2A) gene, achieving a significant reduction in MAT2A expression (P < 0.05) concomitant with the marked inhibition of blastocyst development. Our proposed method, tentatively named ‘Octo-lipofection’, may be useful to analyze gene functions in bovine preimplantation embryos without expensive equipment and skill-intensive techniques.</p>

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