Gene Transfer into Limb Bud of mammalian Embryos Using Electropolation Technique

在人类基因的解码工作基本完成之后，下一步的大课题是基因之间如何相互作用，如何构成复杂的人体结构和功能。本研究采用能完整再现胚胎发育过程的全胚胎培养系统（WEC），建立了小鼠胚胎芽的基因诱导方法，以Std ddt小鼠（E12）为实验材料，在保留约克囊、羊膜、绒毛尿囊胎盘的状态下，解剖出胚胎。将含有GFP的质粒（CMV增强子+β-actin启动子）0.1μl经微毛细管注入约克囊中，注入后立即用钳形元件夹住胚胎，在台氏液中电击（30.40 μ l），然后用荧光显微镜观察。50 V、50 ms、3次脉冲）。然后将胚胎放入瓶中，注入含氨苄青霉素5 μg/ml和葡萄糖2 mg/ml的小鼠血清。旋转瓶在37℃下培养，每天2次通入新鲜的95%O2、5%N2。将旋转瓶以30 rpm旋转。48小时后，取冰冻标本，荧光显微镜下观察，结果如下。在40 V、50 V电压下，电镜下可见GFP在整个植株中表达，而在30 V电压下，仅在芽部可见GFP表达。

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.