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標題: 以 MultiGateway 系統以及 BAC 構築載體並建立 sox-1 knock-in及轉基因之人類胚幹細胞株
Use MultiGateway system and Bacterial Artificial Chromosome to construct vector and establish sox-1 knock-in and transgenic human embryonic stem cell line
作者: 張嘉佑
Chang, Chia-Yu
關鍵字: multigateway;multigateway;BAC;human embryonic stem cell;sox-1;rosette neural stem cell;knock-in;transgene;BAC;人類胚幹細胞;sox-1;rosette neural stem cell;knock-in;轉基因
出版社: 生命科學系所
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在成人的神經系統損傷常會造成嚴重的後果,且因成人體中只有非常少量的神經幹細胞 (neural stem cell) 存在,使創傷後的治療相當困難。近年來胚幹細胞 (embryonic stem cell, ES cell) 的應用正蓬勃的發展, 胚幹細胞擁有可以分化 (differentiation) 為全身各種細胞型態的分化潛力 (pluripotency) 以及在體外培養時自我增生 (self-renew) 的能力,其優秀的自我增生及分化能力被認為是可使用於成體組織修補的最佳素材。藉由最早期神經幹細胞特定表現的細胞標誌 (marker) Sex determining region Y-box 1 (Sox-1) 為鑑定標準分離純化胚幹細胞分化成的神經幹細胞並植入體內修復神經創傷在小鼠模式已經確立其成效。本實驗主要目的以建立sox-1基因敲入 (knock-in) 以及轉基因的人類胚幹細胞為目的,因已經有研究指出人類胚幹細胞體外神經分化時表現的細胞標誌和小鼠胚幹細胞並不相同,其先表現 paired box protein 6 (Pax-6) 而後Pax-6與 Sox-1 同時表現,但其所代表的意義及此時期的細胞接受誘導 (patterning) 分化成為下游神經的能力尚未完全清楚,希望可以以此實驗建立之knock-in或轉基因人類胚幹細胞株在神經幹細胞表現sox-1時期引發的綠色螢光為標準,對此細胞時期更為了解。本實驗各自以不同方法構築了進行knock-in以及轉基因之載體,欲執行knock-in的載體,使用以位點特異性重組(site specific recombination) 原理的three-fragment multiGateway ,此系統只需要一次的重組反應中就能將knock-in所需要的兩段同源性片段 (homologous arm) 以及欲取代人類胚幹細胞染色體上 sox-1 位置的綠螢光及抗生素抗性篩選基因依順序接在knock-in所需要的質體 (plasmid) 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,以大腸桿菌株 EL350 中所含的短片段重組蛋白將 BAC上 sox-1 部分以綠螢光蛋白及抗生素抗性基因取代,此使用 BAC 轉基因的方法主要優點是其所包含的 sox-1 上下游的長片段有相當大的機會包含主要調控 sox-1 表現的區域,較將可能調控 sox-1 表現的區域一一確認並逐步建構轉基因載體更為方便。而後將構築好的knock-in及轉基因載體轉染 (transfection) 進入人類胚幹細胞後建立 sox-1 knock-in及轉基因之人類胚幹細胞株。後續則可以以此細胞株進行 sox-1+ 時期的神經幹細胞的特性確定、其他專一性細胞標誌的尋找以及在體外及體內的分化能力和神經修復能力等測試。

The injury of nervous system leads to serious damage, and still it is difficult to repair the damaged nervous system because of the lack of neural stem cell in adult . The embryonic stem cells (ES cells) have the characteristic of pluripotence and self-renewal , become one of the potentially suitable materials to repair the hurt adult tissue. In the mouse model, Sex determining region Y-box 1 (Sox-1) is one of the earliest neuronal specific cell marker, and the repair efficiency of sox-1 positive neural stem cells differentiated from ES cells in vitro has already been confirmed. The purpose of my experiment is to establish the sox-1 knock-in and transgene human ES cell line. There is remarkable difference of cell specific markers between mouse and human neural stem cells . Previous research suggests that one of the earliest human rosette neural stem cells markers is paired box protein 6 (Pax-6) and at later stage sox-1 express , but the distinguishing features between this two stage are still not really clear now. The sox-1 knock-in and transgene human ES cell line can help us to examine the differentiation potential and specific quality of sox-1+ cell stage of human neural stem cell. In my experience I use two systems to construct the knock-in and transgene vector . The three-fragment multiGateway system can put two homologous arm and the reporter gene needed of knock-in vector together only with two steps site-specific recombination without considering restriction enzyme cutting sites . I use another system to construct sox-1 transgene vector, the bacterial artificial chromosome (BAC) . I choose a human BAC clone that contain sox-1 and it's upstream and downstream sequence that may contain the sox-1 promoter and enhancer region , then we can construct the transgene vector without finding the exactly promoter region. I use a special E.coli strain EL350 that have the ability of short homologous region recombination. We can use this short homologous region recombination system to replace sox-1 region of BAC with fluorescence protein reporter gene . Finally we can transfect these two vectors into human ES cells and establish the sox-1 knock-in and transgene human ES cell line then identify it's characters, differentiation potential and nervous system repair ability.
其他識別: U0005-2107200918032100
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