許多生理現象都需要群體細胞遷移的參與像是形態發生，血管新生以及胚發生。然而，在活體內的群體細胞遷移尚有許多未了解之處。不正常的細胞遷移跟Rap1不正常的表現有關係。黑腹果蠅可以被當做一種模式生物來解開群體細胞遷移之機制。根據先前的研究，邊境細胞的極性在Rap1的持續活化態(Rap1V12)中會被破壞，另一面，在Rap1持續不活化態(Rap1N17)中，細胞突起的數量會減少。這些資料顯示，活化或是不活化態的Rap1對細胞遷移是重要的。但是目前尚未有在活體裡面直接觀測Rap1活性的適當工具。綠色螢光蛋白(GFP)可被用為報導基因。GFP可被拆成三個片段，GFP1-9，GFP10，GFP11，叫做三分子綠色螢光蛋白。可以藉由將二個會有交互作用的蛋白質分別接在GFP10以及GFP11之氮端及碳端，若是一個細胞內同時存在這三個蛋白質，GFP即可重組並發亮。因此，在此利用目前已知可以與活化態Rap1結合的Ras associating domain (RA domain)，標定活化態的Rap1。在此計劃中，三個質體被建構完成，pAc5.1 GFP1-9，pAc5.1 Rap1V12-GFP10，pAc5.1 GFP11-RA1。這三個質體將會被送入果蠅之S2細胞中，測試在細胞內能否使這三個片段重組，並能偵測到GFP，達到標定活化態之Rap1的目的。

A lot of physiological processes, like morphogenesis, angiogenesis and embryogenesis are involved in collective cell migration. However, how does the collective cell migrate in vivo is not well understood. Cell migration defect is affected by abnormally expressing Rap1. Drosophila melanogaster can be used as an animal model to unleash the mechanism of collective cell migration. In the previous study, the polarity of border cell is disrupted in the Rap1 constitutive active system (Rap1V12), on the other side, the protrusion number are decreased in Rap1 dominant negative form system (Rap1N17). These data shown that the active and inactive form of Rap1 is crucial for cell migration. However, there is no appropriate tool to monitor active form of Rap1 in vivo. The green fluorescence protein (GFP) can be used as a reporter. The GFP can be divided into three fragments, GFP1-9, GFP10 and GFP11 which called split GFP. The two proteins which can interact with each other can be fused with N-terminal of GFP10 and C-terminal of GFP11 respectively. The GFP can be reconstituted once these three protein expressed in single cell. Thus, the Ras associating domain (RA domain) which can interact with active form Rap1 are used as a reporter for active form of Rap1. In this project, three constructions, pAc5.1 GFP1-9, pAc5.1 Rap1V12-GFP10 and pAc5.1 GFP11-RA1 have been constructed. These three constructions are going to test if the GFP can be reconstituted and detected or not in Drosophila S2 cell in order to achieve the goal of labeling the active form Rap1.

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