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標題: 以 MultiGateway 系統以及 BAC 構築載體來建立 pax-6 knock-in及轉基因之人類胚幹細胞株
Using MultiGateway system and Bacterial Artificial Chromosome to construct vector for establishment of pax-6 knock-in and transgenic human embryonic stem cell line
作者: 林智堯
Lin, Chih-Yao
關鍵字: 人類胚幹細胞;human embryonic stem cell;基因敲入;轉基因;神經分化;pax-6;knock-in;transgenic;Multigateway;BAC;differentiation;neuron rosette
出版社: 生命科學系所
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胚幹細胞其優秀的自我增生(self-renew)及分化能力(pluripotency)被認為是可使用於成體組織修補的優異再生醫學素材,尤其是再生性較低的神經系統,當神經受損或退化,用細胞治療的方式就相當適合。在人類胚幹細胞的分化研究當中,已有許多探討胚幹細胞分化成神經細胞時期的研究,目前發現在一個分化的特殊時期,具有良好的分化出各式各樣神經細胞的能力,稱為neural rosette,而這個時期所表現的基因為Paired box gene 6 (Pax-6)。本實驗欲建構Pax-6 Knock-in (K-I)人類胚幹細胞株和轉基因 (transgenic)人類胚幹細胞株,希望能以綠色螢光用來代表Pax-6的表現,確立Pax-6基因開始表現的時期以及表現Pax-6的細胞的分化能力研究。本實驗針對欲執行K-I的載體,使用以位點特異性重組(site-specific recombination) 原理的Three-fragment multi-Gateway 系統將K-I所需要的兩段長同源性片段 (homologous arm) 以及欲取代人類胚幹細胞染色體上 pax-6位置的綠螢光及抗生素抗性篩選基因依順序接在 K-I 所需要的質體上。而在轉基因載體的建立,則使用了細菌人工染色體 (bacterial artificial chromosome, BAC) 轉基因系統,以特殊的大腸桿菌株 EL350 中所含的受到溫度調控的重組蛋白,將 BAC上操作 pax-6 部分以綠螢光蛋白及抗生素抗性基因取代。本實驗將構築好的K-I及轉基因載體以 Nucleofector 送入人類胚幹細胞後,以抗生素進行篩選並進行後續鑑定以建立 pax-6 Knock-in及轉基因之人類胚幹細胞株。

Embryonic stem cells (ESCs) can applied to regenerative medicine because their pluripotency and they are good at self-renew. Never system is a low regenesis system, so the ES cell therapy is suitable for repair of injured or degenerated never system. There have been a lot of researches about neural development stage in the human embryonic stem cells (hESCs). One cell type that has the good neuron differentiation ability called neural rosette and paired box gene 6 (Pax-6) is expressed at this stage. We want to establish human pax-6 knock-in and transgenic ES cell line which can be utilized as a model to determine if pax-6 is an appropriate marker for distinguishing the hES differentiation stage. At the beginning, we have constructed knock-in and transgenic vector by different method. We used the three-fragment multi-gateway system which mimics the site specific recombination to make the knock-in vector. To make the knock-in vector, we need to clone upstream and downstream homologous region and make reporter gene, like green fluorescence, and a selection gene. Meanwhile, we search the human bacterial artificial chromosome (BAC) contained pax-6 gene and design a vector to make pax-6 on the BAC can be replaced by green fluorescence gene. The Escherichia coli strain EL350 contains a thermo-sensitive recombinase and it can achieve in vitro homologous recombination. Transgenic vector can use the EL350 system to construct efficiently. We will send Knock-in and Transgenic vector in hESCs by Nucleofector and screen the hES cell line with appropriate antibiotics.
其他識別: U0005-2207200910465600
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