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Precise Manipulation of Developemental transgenic cells using a microwichirned Technology

使用微波技术精确操作发育转基因细胞

基本信息

批准号：
05455005
负责人：
TAMIYA Eiichi
金额：
$ 4.8万
依托单位：
Japan Advanced Institute of Science & Technology (JAIST)
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1994
项目状态：
已结题

项目摘要

In this study, a novel system for gene transfer has been fabricated using micro-machining techniques. Micromachining techniques have been developed for fabricating semiconductor devices. Microelectrodes and can be fabricated onto the glass plate using lithography and vapor deposition techniques.We have used Medaka (Oryzias latipes) as an experimental fish, because it is a suitable material for embryo engineering research based on the following reasons. Compared with mice, fish fertilizes eggs extemally, and there is no need for implant eggs into the uterus of pseudo pregnant animal. Since fish has a transparent egg membrane, both developement and differentiation process are easy to observe.A high electric field induces a hole of lipid bilayr of the cell membranes and results in destroying the egg yolk and a limited yield. Therefore, the electric field should be localized to a certain part of cytoplasm of the cell to increase the yield. Micro-electrodes were fabricated on glass plate with micro-machining techniques to introduce gene to them. The material of the bottom of the electrode is Cr, and the upper part is Au. A polymer film with a hole (1.2mm in the diameter) is attached with an adhesive.Microelectrodes was used for application of localized high electric field at the animal pole of the fertilized egg. High electric field pulses (750 V/cm-1000V/cm) of duration fifty microseconds was applied at a repetition rate of 1Hz for five seconds. Microscopic observation showed no damage of eggs using our system.Forty-one percent of the eggs show luminescence (19 samples in 46 samples). The present system gave a higher ratio of the expression of luciferase than a conventional electroporation method did.The dependence on the cleavage stages about electroporation was measured.Gene transfer was observed in the range from 1-cell stage to 64-cell stage embryo.

在这项研究中，一个新的系统，基因转移已制造使用微机械加工技术。微机械加工技术已经被开发用于制造半导体器件。微电极可以通过光刻和气相沉积技术制作在玻璃板上。我们使用青鳉（Oryzias latipes）作为实验鱼，因为它是基于以下原因的胚胎工程研究的合适材料。与小鼠相比，鱼类在体外使卵子受精，不需要将卵子植入假孕动物的子宫。由于鱼类卵膜透明，发育和分化过程容易受阻，高电场会导致细胞膜脂双层出现孔洞，破坏卵黄，限制产量。因此，电场应局限于细胞质的某一部分，以提高产量。利用微机械加工技术在玻璃板上制作了微电极，用于基因导入。电极底部的材料为Cr，上部为Au。用粘合剂将带有孔（直径为1.2mm）的聚合物膜粘合在一起，用微电极在受精卵的动物极施加局部高电场。以1Hz的重复频率施加持续时间为50微秒的高电场脉冲（750 V/cm-1000 V/cm），持续5秒。显微镜观察表明，使用我们的系统没有损坏鸡蛋。41%的鸡蛋显示发光（46个样品中的19个样品）。结果表明，与常规的电穿孔方法相比，本系统获得了较高的荧光素酶表达率，并测定了电穿孔对卵裂阶段的依赖性，在1-细胞期至64-细胞期胚胎中均观察到了基因转移。