Ikeda Shuntaro, Sugimoto Miki, Kume Shinichi
Laboratory of Animal Physiology and Functional Anatomy, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan.
J Reprod Dev. 2018 Apr 13;64(2):199-202. doi: 10.1262/jrd.2017-137. Epub 2018 Jan 21.
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.
牛植入前胚胎在8 - 16细胞阶段前后表现出显著的生物学变化。在此，我们报告一种简单的脂质体转染方法，利用去除透明带和“孔中孔”（WOW）培养系统将小干扰RNA（siRNA）转染到牛8 - 16细胞阶段的胚胎中。体外产生的牛单细胞胚胎去除透明带后，在WOW培养皿中培养至8 - 16细胞阶段。用siRNA对8 - 16细胞胚胎进行脂质体转染，并在转染48小时后评估转染效率。用红色荧光非靶向siRNA进行脂质体转染揭示了去除透明带对于将siRNA转染到胚胎中的重要性。使用这种方法，我们敲低了甲硫氨酸腺苷转移酶2A（MAT2A）基因，使MAT2A表达显著降低（P < 0.05），同时囊胚发育受到明显抑制。我们提出的这种方法，暂命名为“Octo - 脂质体转染”，可能有助于在无需昂贵设备和技术要求高的情况下分析牛植入前胚胎中的基因功能。
