運用small interfering RNA (siRNA)進行基因沉默(gene silencing)實驗時，抑制基因表現的效率將受到許多因素的影響，包含：細胞與組織接受siRNA的程度、轉錄RNA與蛋白質的半衰期。當研究學者為治療目的而尋求適當的siRNA傳遞方式(siRNA delivery methods)時，報導子(reporter)將成為評估基因沉默實驗的有效工具。在這個研究裡，我們將證實光轉變螢光蛋白(photoconvertible fluorescent protein) Kaede可作為基因沉默實驗上有效的報導子。相較於在傳統上要等待報導子進行自然分解後才能去偵測新生蛋白質生成的速度，在這個實驗裡，我們將在進行RNA干擾(RNA interference)實驗之前，先將Kaede蛋白進行綠色轉成紅色螢光的光轉變反應來減少綠色螢光背景(green fluorescence background)強度，螢光背景的下降將有助於提升在RNA干擾下新生Kaede蛋白的偵測靈敏度。從我們的實驗當中可發現，在12小時的時間點，控制組(scramble)與實驗組(Kaede)的螢光強度相差2.1倍。相較於Kaede，以EGFP作為報導子時，其螢光強度與控制組(scramble)僅相差0.4倍。由實驗結果可證實，以光轉變螢光蛋白Kaede作為報導子時，將可成為偵測RNA干擾機制與siRNA傳遞效率的實用工具。

The efficiency of small interfering RNA (siRNA)-mediated gene silencing is highly variable. The process depends on a number of factors including the efficiency of siRNA uptake by cells/tissues as well as the half-lives of the RNA transcript and the target protein. While investigators are searching for suitable siRNA delivery methods for therapeutic purposes, reporter proteins are typical used to evaluate the result of gene silencing. In the present study, we demonstrate that the photoconvertible fluorescent protein, Kaede, can serve as an effective reporter to study gene silencing. Unlike the conventional approach of waiting for the natural turnover of reporters, we actively induced the green-to-red conversion of Kaede to erase the residual reporter activity prior to siRNA administration. The low residual background led to an increase in sensitivity of detecting the de novo synthesis of Kaede, which served as an effective indicator of gene silencing. Using this approach, we were able to initiate a significant 2.1-fold difference in fluoresce signals between the Kaede-specific and scramble siRNAs transfected cells twelve hours post administration. In comparison, only 0.4-fold difference was observed while using GFP as the reporter. Our work demonstrates a new application of photoconvertible fluorescent protein Kaede as the gene-silencing reporter, which can be a useful tool for monitoring the mechanism of RNA interference and assessment of appropriate methods for siRNA delivery.

中文摘要 2
Abstract 3
縮寫與全名對照表 5
謝誌 6
目錄 7
圖表目錄 9
第一章 文獻回顧 10
1-1基因沉默(gene silencing) 10
1-2 siRNA傳遞方式(delivery of siRNA) 13
1-3報導基因(reporter gene) 15
1-4光轉變螢光蛋白-Kaede
(photoconvertible fluorescent protein -Kaede) 20
1-5 Kaede和EGFP的比較 22
1-6提昇轉染效率藥劑：氯喹(chloroquine) 23
1-7實驗目的 25
第二章 材料與方法 26
2-1人類纖維細胞HT-1080 (human fibroblasts HT-1080) 26
2-2重組基因質體 26
2-3共同轉染(co-transfection) 29
2-4挑選含有Puromycin resistance的螢光細胞 30
2-5使用紫外光照射細胞 31
2-6細胞活性分析(cell viability assay) 33
2-7 RNA干擾 (RNA interference , RNAi) 35
2-8提昇轉染效率藥劑：氯喹(chloroquine) 38
第三章 實驗結果與討論 41
3-1單株細胞篩選 41
3-2紫外光照射細胞 41
3-3紫外光照射的時間 42
3-4 RNA干擾(RNA interference，RNAi)實驗 43
3-5提昇轉染效率藥劑：氯喹(chloroquine) 45
第四章 結論 59
第五章 未來展望 61
參考文獻 63

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