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論文中文名稱:電擊法對轉染效率及拓樸異構酶 IV對質體長期表達之研究 [以論文名稱查詢館藏系統]
論文英文名稱:The effects of electric-treated DNA-liposome complex on transfection efficiency and topoisomerase IV for plasmids long term expression in CHO cells [以論文名稱查詢館藏系統]
院校名稱:臺北科技大學
學院名稱:工程學院
系所名稱:生化與生醫工程研究所
畢業學年度:101
出版年度:102
中文姓名:林廷陽
英文姓名:Ting-Yang Ling
研究生學號:100688012
學位類別:碩士
語文別:中文
口試日期:2013-07-26
論文頁數:52
指導教授中文名:侯劭毅
口試委員中文名:黃光策;吳宛儒;翁文慧
中文關鍵詞:綠色螢光蛋白拓樸異構酶IVLipofectamine 2000
英文關鍵詞:EGFPTopoisomerase IVLipofectamine 2000
論文中文摘要:目前動物細胞的研究,已有許多種不同載體的轉染方式,包括利用病毒性載體(例如腺病毒、反轉錄病毒)、物理性基因傳遞(電擊法)以及市面上商業類轉染試劑等幾種方法。雖然以病毒當作載體進行實驗,可以得到較高的轉染效率,不過病毒的DNA會崁入細胞的染色體。為了避免載體的 DNA 崁上細胞染色體,所使用的載體改以質體進行實驗。以誘導型幹細胞為例,使用環形質體當作載體的時候會使轉型成誘導型幹細胞的效率下降許多,及轉染後成功帶有質體的細胞需要能長期表達等限制。此外有文獻指出影響轉染效率有很多因子,如DNA-liposome的比例、質體的純度及粒徑大小等。
在本實驗中,我們以在中國倉鼠卵巢細胞(CHO k1 cell)能表現綠色螢光蛋白質的pBIG ( EGFP ) 作為表現用質體,及具有拓樸異構酶 IV 的質體pCIE並使用Lipofectamine 2000 作為攜帶這兩個質體的轉染試劑( parC/parE ),觀察外來質體是否在細胞內長期表達,另一方面利用物理性電擊法觀察DNA-liposome complex的粒徑是否經過電擊而發生變化及未電擊及電擊後的lipoplexes是否會影響轉染效率。實驗結果顯示,帶有拓樸異構酶IV的細胞對外來質體的長期表達沒有太大幫助,另一方面經過電擊後處理的轉染效率較未電擊的轉染效率差,且DNA-liposome complex的粒徑經電擊處理會變大。
論文英文摘要:Transfection techniques of animals cell, include the use of viral vectors, physical gene delivery methods, and commercial transfection reagents.Although viral vectors have high transfection efficiency, their DNA can also integrate into the chromosome of the host cell. On the other hands, the used plasmid vector could avoid integrating vector DNA into the chromosome. For example, the induction efficiency of iPS cells is very low and needs longer expression times of reprogramming factors. Many factors affect transfection efficiency of the transfection process. For example, as the size of liposome particles and DNA-liposome complex increases, the transfection efficiency also increases.
We tested the transfection efficiency by two plasmids: one encoding EGFP named pBIG that could produce green fluorescent protein in mammalian cells and the other encoding parC/parE that could produce topoisomerase IV named pCIE and Lipofectamin 2000 as transfection reagents to observe. We demonstrate that the size of DNA-liposome complex increases after electric shock; however, the transfection efficiency decreased.
論文目次:摘要 i
誌謝 iii
目次 iv
表目錄 vi
圖目錄 vii
第一章 緒論 1
1.1 前言 1
1.2 研究目的 2
第二章 研究背景與原理介紹 3
2.1 微脂體之簡介 3
2.1.1 陽離子型微脂體(Cationic liposome) 3
2.1.2 陽離子型微脂體的轉染機制 4
2.1.3 影響陽離子微脂體轉染效率的因子 5
2.2 MTT assay 6
2.3 誘導型幹細胞 7
2.4 拓樸異構酶 9
2.4.1 第一型拓樸異構酶 9
2.4.2 第二型拓樸異構酶 9
第三章 研究方法 11
3.1 電擊法對轉染效率影響之實驗架構 11
3.2 拓樸異構酶IV在細胞裡對質體維持影響之實驗架構 12
3.3 質體pBIG、pCIE 的純化 12
3.4 CHO k1 細胞培養(中國倉鼠卵巢細胞) 14
3.4.1 冷凍細胞活化 14
3.4.2 細胞繼代 15
3.4.3 冷凍細胞 16
3.4.4 細胞計數 16
3.5 轉染方法 17
3.5.1 轉染前一天 17
3.5.2 轉染當天 18
3.6 螢光顯微鏡觀察細胞型態及轉染效率 20
3.7 MTT assay 20
3.8 質體 pCIE、pBIG 共同轉染 21
3.9 螢光顯微鏡觀察後之繼代 21
3.10 質體 pCIE、pBIG繼代後之螢光觀察 22
3.11 實驗藥品 23
3.12 實驗設備 25
第四章 電擊法對轉染效率的影響 27
4.1 轉染效率分析 27
4.1.1 固定DNA用量調整LF2000比例 27
4.1.2 固定LF2000用量調整DNA比例 29
4.2 細胞毒性分析 30
4.2.1 固定DNA用量調整LF2000比例 30
4.2.2 固定LF2000用量調整DNA比例 32
4.3 粒徑大小檢測 33
4.3.1 固定DNA用量調整LF2000比例 33
4.3.2 固定LF2000用量調整DNA比例 34
第五章 拓樸異構酶IV在中國倉鼠細胞內對質體的影響 36
5.1 轉染pCIE後對質體的影響 36
5.2 轉染後之細胞doubling time 37
第六章 結論 39
參考文獻 41
附錄 43
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