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標題: 利用一個新技術去快速建立DF-1及NIH 3T3穩定細胞株
A novel strategy of establishing an in vitro assay system in DF-1 and NIH3T3 cell lines
作者: 毛敏驊
Mao, Ming-Hua
關鍵字: 穩定細胞株;cre
出版社: 生命科學系所
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當細胞受到外來刺激時,會產生許多機制去做不同的調節,通常會需要合成新的mRNA以及蛋白質。在mRNA的層級,對於調節細胞的反應情形可以由Real-time PCR(即時連鎖聚合酶反應)來得知訊息。然而這些結果卻無法應用在轉譯前或轉譯的蛋白質上面。為了要能得知細胞如何去反應外來訊息時,而且必須能在蛋白質上得到正確地以及並可重複性地定量蛋白質的量與其活性。但是現今發現一般短暫的轉染作用並沒有辦法能夠去精準分析測出轉錄因子的活性。因此我們想要在in vivo或是在in vitro中發展出一個平台利用結合Cre/loxP系統與雙冷光酵素分析系統作為分析定量出轉錄因子的活性或數目。為了將細胞的變化值減到最小,我們設計了帶有firefly luciferase reporters穩定細胞株的建立,並且利用fusion protein(同時帶有Renilla luciferase與轉錄因子構築融合蛋白(fusion protein))去計算出單獨轉錄因子的數量。為了要能縮短建立多種穩定細胞株的時間,因此我們使用Cre/loxP系統能有效做目標序列的轉換。我們分別利用DF-1與3T3這兩株細胞株,去建立帶有reporters的穩定細胞株,期望其細胞株中帶有的lox sites能夠快速地將目標序列進行置換。更進一步地,希望這個系統能夠被應用於不同的蛋白質在轉殖動物中的功能。

To react to extracellular stimuli, cells employ various mechanisms to regulate different cellular responses, which usually require synthesis of novel mRNAs and proteins. Real-time PCR has provided valuable quantitative information on the regulation of cellular responses at mRNA level; however those results are not applicable to proteins that have translational or post-translational control. To obtain accurate and reproducible quantitative information on how cells respond to extracellular signals at protein level, we found the conventional transient transfection strategy that can not precisely measure transcription factors activity is not sufficient. Therefore, we intend to develop a platform that combines Cre/loxP system and dual luciferase reporter system for quantitative analysis of transcription factors activity in vitro and in vivo. To minimize cellular variations, we generated stable cell lines for firefly luciferase reporters, and used Renilla luciferase-fused transcription factor to measure the quantity of individual transcription factor. Also to facilitate the time-consuming processes of generating multiple stable cell lines, we used Cre/loxP system for efficient target sequence swapping. To demonstrate the practical applications of this strategy, we generated a collection of reporter cell lines from DF-1 and NIH/3T3 cells containing lox sites for rapid exchange of target DNA sequences. Further, this system can be applied to study the function of different proteins in transgenic animals.
其他識別: U0005-2901200809240800
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