The development of photoconversion fluorescent protein (PCP) assisted-fluorescence microscopic technique for single cell gene expression analysis

[[abstract]]本實驗構想為利用顯微螢光技術觀測單一細胞之生理活性。所探討的問題是以前因未曾使用此技術而較少被提及或較困難討論的調控基因表現順序問題。此實驗使用之偵測關鍵技術為利用光轉換螢光蛋白的特殊特性。藉由共軛焦顯微鏡的觀察下，當細胞接受刺激而進行反應時，特定啟動子會調控其下游基因進行表現。若將光轉換螢光蛋白基因構築於這些特定基因下游，藉由螢光蛋白之量測，即可推知啟動子活性與所接受刺激間之相互關係。但原先接受刺激前所產生之螢光蛋白會影響後來實驗結果，因此這些光轉換螢光蛋白經激活光照射後，會進行不可逆的螢光轉換(由無螢光變為有螢光或改變螢光放射波長)，即可克服此問題。本實驗所使用之光轉換螢光蛋白(Dendra2及PSmOrange)即可由原本之綠色螢光轉成紅色螢光及橙色螢光轉成遠紅螢光。如此便能區分刺激前後啟動子活化程度，進而探討重要細胞生理活性進行時，其中相關啟動子間相互之活化順序、強度、及交互作用。當細胞接受刺激後，基因啟動子需表現下游基因以平衡外來刺激。因此量測基因啟動子所需之時間即為一重要課題。待測知基因表現率後，接續之基因表現相互關係即可定義。以及加入藥物後，針對訊號傳遞上游、下游之間的相互關係是否會因為藥物加入的影響，而導致促進訊號傳遞的速率。[[abstract]]The aim of this proposal is to develop the fluorescence microscopic technique to explore the genetic activities within single cells and living tissues. The questions asked in this proposal are the promoter-controlled gene expression sequences which are less to be discussed or hard to be answered due to the absence of this technique. The key techniques of this proposal are the use of photo-conversion fluorescent proteins (PCP), for example, Dendra2 and PSmOrange; and the confocal microscopic analysis. Once activated by environmental stimuli, promoters will express the downstream genes to respond the stimuli. If PCP genes are constructed under the control of specific promoters, the measurement of the expression of PCPs is then able to deduce the activation level of promoters. However, the initially expressed PCPs may interfere with the measurement. Thus, one of the merits of PCPs is their photo-conversion property. The emission wave length of PCPs is varied after activating irradiation. This may help ”reset” the protein expression in the cells. Dendra2 and PSmOrange, a PCP used in the proposal, may undergo emission wave length alteration and have a fluorescence change from green to red. Accordingly, the measurement of the newly synthesized green Dendra2 can distinguish these proteins from those initially produced ones (have already changed to red fluorescence) and orange PSmOrange have already changed to far-red fluorescence. And after treating drugs, whether the drugs to influence signal transduction pathway relationship, result in improve the signal transduction rate.[[note]]碩