MOLECULAR MECAHNISMS OF ENDODERM DIFFERENTIATION

通过使用胚胎干（ES）细胞，基因陷阱现在可以在小鼠中实现。该方法利用含有剪接受体、报告基因和质粒DNA的基因诱捕载体，具有分离未知基因、破坏未知基因以产生敲除小鼠和分析未知基因表达模式三个优点。然而，筛选系统的选择是有效识别未知基因的关键。我们利用胚状体的形成建立了新的筛选体系。囊状胚状体具有相似的内胚层标记基因表达模式，从而允许胚状体形成作为筛选系统。当载体整合到串联阵列中时，难以分离5'侧翼区。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，通过Cre的瞬时表达可以去除载体的额外拷贝。利用该系统共分离出120个ES诱捕克隆，并根据它们在胚状体形成过程中标记基因的表达模式将它们分为6组。从四个诱捕克隆中分离5'侧翼区并测序。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敲除小鼠方面是相当有效的。

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.