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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技术中最耗时、最费钱的环节.如果没有这一步，转基因必须更有效地进行。本课题的目的是研究酪氨酸酶小基因作为转基因中可见报告基因的可能性。如果成功的话，我们就可以消除使用从转基因候选动物的组织中提取的基因组DNA进行转基因检测，这是转基因中必不可少但最耗时和最费钱的步骤。报道基因的条件是：1）其在真实组织如皮肤和眼睛中的毛囊中稳定表达，2）尽可能小的尺寸，因为报道基因必须与其他感兴趣的基因连接。为了检验这些方面，我们构建了一个新的酪氨酸酶小基因mg-Tyrs-JS，其启动子区截短，比Tanaka等（1990）构建的小基因mg-Tyrs-J缩短了1.9Kbp。采用电穿孔法将mg-Tyrs-Js小基因导入小鼠L细胞，获得了mg-Tyrs-Js小基因的表达。然后，我们用这个小基因制造了转基因小鼠。然而，出乎意料的是，通过我们的努力，没有获得携带该小基因的转基因小鼠，这表明在被消除的启动子区域中必须存在其他组织特异性调控元件。