Precise Manipulation of Developemental transgenic cells using a microwichirned Technology

点击翻译按钮获取中文摘要

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.