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研究生:林智堯
研究生(外文):Chih-Yao Lin
論文名稱:以MultiGateway系統以及BAC構築載體來建立pax-6knock-in及轉基因之人類胚幹細胞株
論文名稱(外文):Using MultiGateway system and Bacterial Artificial Chromosome to construct vector for establishment of pax-6 knock-in and transgenic human embryonic stem cell line
指導教授:蘇鴻麟蘇鴻麟引用關係
指導教授(外文):Hong-Lin Su
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生命科學系所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
畢業學年度:97
語文別:中文
論文頁數:74
中文關鍵詞:人類胚幹細胞基因敲入轉基因神經分化
外文關鍵詞:human embryonic stem cellpax-6knock-intransgenicMultigatewayBACdifferentiationneuron rosette
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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.
目錄
中文摘要 i
英文摘要 ii
圖表目錄 iv
第一章、 前言 1
第二章、 材料與方法 11
第三章、 結果 28
第四章、 討論 35
第五章、 參考文獻 38
圖表 43
附錄 73

圖表目錄
圖1.構築Venus-NLS 43
圖2.構築篩選基因loxP2Pgkβ-geo 44
圖3.構築Venus-NLS_loxP2Pgkβ-geo 45
圖4.確認VenusNLS的表現 46
圖5.Gateway 三段PCR 47
圖6.上下游片段BP reaction 48
圖7.Knock-in Insert片段BP reaction 49
圖8.構築pDEST R4R3 將上負篩選基因Pgk-DTA 50
圖9.建構欲進行Pax-6轉基因載體流程示意圖 51
圖10.設計欲進行短片段重組之質體示意圖 52
圖11.Pax-6上游同源片段5’ flanking region選殖 53
圖12. Pax-6下游同源片段3’ flanking region選殖 54
圖13.報導基因VenusNLS的選殖 55
圖14.確認報導基因VenusNLS的表現 56
圖15.構築PL452-Pax6 BACTG 3''flanking 57
圖16.構築PL452-Pax6 BACTG 3''flanking-5''fanking 58
圖17.構築PL452-5’-3’-VenusNLS 59
圖18.確認RP11-804L22電穿孔到EL350 60
圖19.將PL452-5’-3’-VenusNLS剪切成線狀 61
圖20.確認Pax-6 BAC重組成功 62
圖21.將重組的Pax-6 BAC 以限制酵素確認 63
圖22. 將TE671 cDNA以PCR確認pax-6 表現 64
圖23.繼代中之人類胚幹細胞在顯微鏡觀察下的型態 65
圖24.神經分化流程示意圖 66
圖25. EB 生成 67
圖26.人類胚幹細胞進行神經分化後Neural Rosette之構造 68
圖27.人類胚幹細胞進行 single cell dissociation之細胞型態 69
表1.建構knock-in之引子 70
表2.建構transgene之引子 71
表3.確認Pax-6 cDNA表現之引子。 72
附錄圖1. Multi Gateway 流程示意圖 73
附錄圖2. 藉由重組交換將BAC 序列片段轉殖到載體 74
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