Gene Transfer into Limb Bud of mammalian Embryos Using Electropolation Technique

使用电插技术将基因转移到哺乳动物胚胎的肢芽中

• 批准号: 15591905
• 负责人: KISHI Yoko
• 金额: $ 2.24万
• 依托单位: Jikei University School of Medicine
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15591905/
• 关键词: Gene transfer; The whole embryo culture system; Electropolation; エレクトロポレーション法による遺伝子導入; 四肢の発生

After the decording of human gene was almost finished, Next big theme is how each gene interrlate to the other genes, and how to make the complex human structure and function. In this study, the whole embryo culture(WEC)system which is able to reproduce the development completely has been used, we talk about the establishment of the way to induce gene to the mouse embryo bud.Using Std-ddt mouse(E12), Embryo was dissected out at the state of remaining of the york sac, amnion, chorioallantoic placenta. Plasmid(CMV enhancer+β-actin promoter) added GFP 0.1μl was injected by microcapillery tube into the york sac Immediately after injection of material, the embryo was pinched by the pincet type element.In the Tyrode's solution Electric shock(30.40. and 50V,50ms,3pulse) was applied to the embryo. Then Embryo was put into the bottle within which the mouse serum with ampicirin 5 μg/ml and glucose 2mg/ml was infused. The rotating bottles were incubated at 37℃, fresh 95% O2,5% N2 was supplied to the bottle at 2 times a day. The rotator bottle were turened at 30rpm. After 48 hours the frozen sample was resected, we observated them by fluoscent microscope.Below was the results. At the power of 40v,50v, the GFP was detected in the whole body, but at the power of 30v the only bud portion the GFP was observed by the electric microscope.

在人类基因的解码基本完成之后，下一个大的主题是每个基因如何与其他基因相互关联，以及如何使复杂的人类结构和功能。本研究采用能够完全再现发育的全胚培养（WEC）系统，探讨建立将基因诱导至小鼠胚芽的方法。采用Std-ddt小鼠（E12），在卵囊、羊膜、绒毛膜和尿囊胎盘残馀状态下取出胚胎。将添加GFP 0.1μl的质粒（CMV增强子+β-肌动蛋白启动子）通过毛细管注入约克囊，注射后立即用针型元件捏胚。在Tyrode溶液中电击（30.40）。50V,50ms，3脉冲)。然后将胚胎放入瓶中，瓶中注入含氨苄青霉素5 μg/ml和葡萄糖2mg/ml的小鼠血清。旋转瓶在37℃，新鲜95% O2,5% N2中孵育，每天2次。旋转瓶以30rpm旋转。48小时后，取冷冻标本，用荧光显微镜观察。结果如下：在功率为40v、50v时，可以检测到全身的绿色荧光蛋白，而在功率为30v时，显微镜只观察到芽部的绿色荧光蛋白。