Establishment of Transgene Detection System with Mouse Tyrosinase Gene as a Visible Reporter Gene

以小鼠酪氨酸酶基因为可见报告基因的转基因检测体系的建立

基本信息

批准号：
05680747
负责人：
YONEKAWA Hiromichi
金额：
$ 1.22万
依托单位：
The Tokyo Metropolitan Institute of Medical Science
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
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

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05680747/
关键词：
Tyrosinase Transgenesis Visible Marker Transgene Genotoxicity Gene Manipulation

项目摘要

The transgene detection is the most time- and money- consuming step in transgenesis. Without this step, transgenesis must be proceeded more efficiently. The aim of our project is to examine the possibility whether tyrosinase minigene can be used to be a visible reporter gene in transgenesis. If succeeded, we can eliminate the transgene detection using genomic DNA extracted from tissue of transgenic candidate animals, which is essential but the most time- and money-consuming step in transgenesis. The conditions for the reporter gene are : 1) its stable expression in authentic tissues such as hair follicle in skin and eye, 2) minimum size as possible because that the reporter gene must be connected with other gene (s) of interest. For examine these respects, we have constructed a new tyrosinase minigene mg-Tyrs-JS with its truncated promoter region, 1.9 Kbp shortened than the previous minigene mg-Tyrs-J constructed by Tanaka et al. (Devel-opment 00 : 000-000,1990) . Introduced into cultured mouse L cells by electroporation method, the truncated minigene mg-Tyrs-Js was expressed in the cells. Then, we made transgenic mice using this minigene. Unexpectedly, however, no transgenic mice carrying with this minigene could be obtained with our great efforts, suggesting that the other tissue-specific regulatory element (s) must exist in the eliminated promoter region.

转基因检测是转基因中最耗时和赚钱的步骤。没有此步骤，必须更有效地进行转基因。我们项目的目的是检查酪氨酸酶微糖是否可以用作转基因中可见的记者基因的可能性。如果成功，我们可以使用从转基因候选动物组织中提取的基因组DNA消除转基因检测，这是转基因中最重要的和耗资最耗费的步骤。记者基因的条件是：1）其在皮肤和眼睛中毛囊等正宗组织中的稳定表达，2）尽可能最小的大小，因为报告基因必须与其他感兴趣的基因相连。为了检查这些方面，我们构建了一个新的酪氨酸酶Mimigene Mg-Tyrs-J，其截短的启动子区域比田中等人构建的先前的微型MG-TYRS-J缩短了1.9 kbp。（Devel-opment 00：000-000,1990）。通过电穿孔方法引入培养的小鼠L细胞，在细胞中表达了截短的微酸MG-TYRS-JS。然后，我们使用这种微基制成了转基因小鼠。然而，出乎意料的是，我们的巨大努力可以获得不带这种微基因的转基因小鼠，这表明在被淘汰的启动子区域中必须存在其他组织特异性的调节元件。