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MOLECULAR MECAHNISMS OF ENDODERM DIFFERENTIATION

内胚层分化的分子机制

基本信息

批准号：
07457555
负责人：
YAMAMURA Kenichi
金额：
$ 4.74万
依托单位：
KUMAMOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07457555/
关键词：
ES CELL GENE TRAP EMBRYOID BODY

项目摘要

Through the use of embryonic stem (ES) cells, the gene trap are now possible in the mouse. In this approach we use the gene trap vector which contains splice acceptor, reporter gene and plasmid DNA.This approach has three advantages, the isolation of an unknown gene, disruption of its unknown gene leading to the creation of knock out mouse, and the analysis of expression pattern of its unknown gene. However, it is essential to choose the screenig system to identify an unknown gene efficiently. We developed the new screenig system using the embryoid body formation. Cystic embryoid body was shown to have similar expression patterns of endodermal marker genes, thus allowing the embryoid body formation as a screenig system. It is difficult to isolate the 5' falnking region when the vector was integrated in an tandem array. To overcome this problem, we used the Cre-loxP system. We constructed the new trap vector which contains two loxP sites. It was shown that the extra copies of vectors can be removed by the transient expression of Cre. Using these system we isolated 120 ES trap clones and divided them into 6 groups according to thier expression pattern of marker gene during embryoid body formation. The 5' flanking region was isolated from four trap clones and sequenced. Three of them were known genes and the other was unknown, suggesting that this method is quite efficiente in defining unknown genes and production of knock out mouse.

通过使用胚胎干细胞(ES)，现在可以在小鼠身上捕获基因。在该方法中，我们使用了包含剪接受体、报告基因和质粒DNA的基因陷阱载体，该方法具有三个优点：分离未知基因，干扰其未知基因导致基因敲除小鼠的产生，以及分析其未知基因的表达模式。然而，选择Screenig系统来有效地鉴定未知基因是必不可少的。我们开发了利用类胚体形成的新的筛选系统。囊状胚状体具有相似的内胚层标记基因表达模式，从而使胚状体形成为一个筛选系统。当载体整合到串联阵列中时，很难分离出5‘端错配区域。为了克服这个问题，我们使用了CRE-loxP系统。我们构建了含有两个loxP位点的新的陷阱载体。结果表明，Cre的瞬时表达可以去除载体的多余拷贝。利用该体系分离了120个ES诱捕克隆，并根据标记基因在类胚体形成过程中的表达模式将其分为6组。从4个陷阱克隆中分离出5‘侧翼区，并对其进行了序列测定。其中3个为已知基因，1个为未知基因，表明该方法在确定未知基因和生产基因敲除小鼠方面是非常有效的。