MOLECULAR MECAHNISMS OF ENDODERM DIFFERENTIATION

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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.