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

The effects of electric-treated DNA-liposome complex on transfection efficiency and topoisomerase IV for plasmids long term expression in CHO cells

林廷陽(Ting-Yang Ling)

指導教授 ： 侯劭毅

國立臺北科技大學/工程學院/生化與生醫工程研究所/碩士(2013年)

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。

關鍵字

綠色螢光蛋白 ； 拓樸異構酶IV ； Lipofectamine 2000

並列摘要

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.

並列關鍵字

EGFP ； Topoisomerase IV ； Lipofectamine 2000

參考文獻

24.黃采瑜，經電擊處理後之微脂體對轉染效率的影響之研究。(2012)，台北

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