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

转基因检测是转基因过程中最耗时、最费钱的一步。如果没有这一步，转基因必须更有效地进行。本课题的目的是探讨酪氨酸酶基因作为转基因报告基因的可能性。如果成功，我们可以利用从转基因候选动物组织中提取的基因组DNA来消除转基因检测，这是转基因过程中必不可少的，但也是最耗时和最耗资的步骤。报告基因的条件是：1)在皮肤和眼睛的毛囊等真实组织中稳定表达；2)报告基因必须与其他感兴趣的基因(S)连接，因此大小尽可能小。为了检验这些方面，我们构建了一个新的酪氨酸酶微型基因MG-Tyars-JS，它的启动子区域被截短，比Tanaka等人构建的微型基因MG-Tyars-J缩短了1.9kbp。(发展--1990年00：000-000)。用电穿孔法将截短的微小基因MG-TyrS-Js导入培养的小鼠L细胞，并在细胞内表达。然后，我们用这种微型基因制造了转基因小鼠。然而，出乎意料的是，在我们的努力下，没有获得携带该微型基因的转基因小鼠，这表明在消除的启动子区域中肯定存在另一个组织特异性调控元件(S)。

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.