新穎技術之建立-利用TALEN技術建構斑馬魚基因敲除模型及新艷紅染料與石墨烯之修飾電極製備及對去氧核醣核酸鹼基電催化之研究

Development of new technology to efficient TALEN-mediated gene knockout in zebrafish and detection of DNA Bases on electrodes chemically modified with graphene-new fuchsin

陳佳怡(Jia-Yi Chen)

指導教授 ： 翁文慧

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

TALEN 是基因組修飾工具，透過重複序列辨識 DNA 序列，再由 FokI 內切酶來剪切雙股DNA，造成雙股斷裂達成基因剔除的目的。本研究先利用螢光原位雜合技術探討基因表現，發現多組細胞株都有TNFAIP3缺失之現象。因此，我們使用TALE 核酸酶技術建構出TNFAIP3基因定點敲除之斑馬魚突變模型，進一步研究A20基因在脊椎動物胚胎發育的功能，檢視缺失TNFAIP3基因所造成的疾病表徵，並利用此斑馬魚株來研究類風濕性關節炎、牛皮癬和淋巴癌等免疫疾病的病理機制，將來並可幫助進一步用作日後藥物篩選的平台。
本論文另一研發目標為利用石墨稀與新豔紅聚合物結合形成複合膜，並以定電位的方式將膜修飾在玻璃碳電極上藉石墨稀存在提高了表面覆蓋濃度、增加電和轉移速率常數（Ks）並且減少新豔紅高分子在多迴圈長膜中的損失。實驗結果也顯示出此複合薄膜對腺嘌呤、鳥嘌呤、胸腺嘧啶和胞嘧啶這些生化物質的混合物有增加電催化活性的發展性。而附和性薄膜的表面型態，是將其製備在透明的ITO導電玻璃上，在利用掃描式電子顯微鏡以及原子力電子顯微鏡來進行觀察。由這兩種儀器可以觀察到新豔紅聚合與石墨稀結合在玻璃碳電極上，對於複合膜的電催化訊號分析是以微分脈衝伏安法和循環伏安法來進行測量，DPVs不僅增加測量的電流之線性濃度範圍，也降低過氧化電位的干擾。而石墨稀發展至今,深具實際應用的潛力與增強對分析物的電催化活性提高負載和穩定。因此，在本文中，利用此複合膜催化胞嘧啶（C），胸腺嘧啶（T），鳥嘌呤（G），腺嘌呤（A）來分析催化訊號。

關鍵字

TNEAIP3 ； TALEN ； A20 ； HRM薄 ； 膜修飾電極 ； 新艷紅 ； 電催化 ； 胞嘧啶 ； 胸腺嘧啶 ； 鳥嘌呤 ； 腺嘌呤

並列摘要

TALEN nuclease (TALENs) is another new technology to specifically and effectively modify genome. The DNA binding domain of TALENs fused to the FokI cleavage domain can be used generate chimeric nuclease (TALENs) that bind to DNA and create double strand breaks. In the present study, we have collected cell line, and new, molecular biology techniques including fluorescence in situ hybridization (FISH) was used to investigated the aberrations of genes TNFAIP3 in all cell line. In previous study, the deletion of gene A20 in several cell lines was observed by fluorescence in situ hybridization (FISH). We aimed to explore the biological function of A20 by knock-out A20 gene function by using TALEN technology.The A20 knockout zebrafish established in this study provide a precious and simple animal model to ask the potential function of A20 in vertebrates.

We report the fabrication of Graphene combined with New Fuchsin (NF) to prepare Graphene-NF modified electrode. Graphene obtained high mechanical strength, non-antigenic biopolymer and low toxicity toward mammalian cells. Electrochemical impedance spectroscopy (EIS) applied diffusion coefficient values and some information about the kinetics of electron transfer during the redox reactions. Surface morphology of the modified electrode using scanning electron microscopy (SEM) and atomic force microscopy (AFM), which revealed that Graphene and NF were coated on electrode. Differential pulse voltammetry (DPVs) was used for the determination of analytes. DPVs not only increased the electrocatalytic current linear concentration range, also lowered the overpotential to oxidation the interferences in the measurements. The Graphene exhibited a promising enhanced electrocatalytic activity towards analytes and enhances the loaded and stability. In this paper, the electrochemical oxidation of Cytosine (C), Thymine (T), Guanine (G) and Adenine (A) at same time.

並列關鍵字

TNEAIP3 ； TALE ； HRM ； Modified electrode ； New Fuchsin ； Cytosine (C) ； Thymine (T) ； Guanine (G) ； Adenine (A)

參考文獻

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