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研究生:洪再賢
研究生(外文):Tsai Hsien Hung
論文名稱:建立快速簡便的反轉錄病毒載體系統技術平台
論文名稱(外文):Establishment of a Convenient Retroviral Vector Technical Platform
指導教授:張國友
指導教授(外文):K. Y. Chong
學位類別:碩士
校院名稱:長庚大學
系所名稱:醫學生物技術研究所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
論文頁數:96
中文關鍵詞:慢病毒載體Gateway 位點特異性重組系統微型核糖核酸啟動子
外文關鍵詞:lentiviral vectorgateway recombination systemmicroRNApromoter
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慢病毒為反轉錄病毒科中的一屬,其具有可以感染分裂細胞和非分裂細胞的能力,由於慢病毒可以高效率的把基因轉染到哺乳動物細胞中,所以被當作基因轉移工具應用於基因治療研究領域。然而傳統的基因選殖技術常因找不到合適的限制酶切位及接入基因片段方向性等問題使得實驗變得複雜且費時。近來科學家研發一種新型的基因重組技術稱為「Gateway位點特異性重組系統」,主要利用載體上特異性位點間的重組來完成基因片段與載體之間快速簡便的互換。此系統不僅可以省略使用限制酶和連接酶,甚至可以確保基因閱讀框的方向正確性。因此本實驗希望利用Gateway位點特異性重組系統建構多種快速且簡便的反轉錄病毒載體技術平台,因此我們的研究著重於探討慢病毒載體基因表現效率,以及研發快速簡便的方法將基因片段送入慢病毒載體系統進行表達。為了應用在不同之基因治療上,啟動子(Promoter)的選擇將會影響所攜帶之基因的表現。目前有很多以RNA polymerase II 系統為基礎之不同的啟動子,其中包括持續表現基因的CMV、UBC promoter 及可調控表現基因CMV-TO,這二類三種的啟動子各有不同之特性:如Human cytomegalovirus (CMV) promoter,為病毒性的啟動子之一,它能在大部分的哺乳類動物細胞中持續驅動攜帶基因的表現;Human ubiquitin C (UBC) promoter,為內源性的啟動子且因ubiquitin C基因被高度的保留在所有真核細胞中,所以能夠在哺乳類動物的細胞中被誘發及持續表現基因。另外, CMV-TO promoter是建立在tetracycline repressor(TetR)可調控系統上的啟動子,Escherichia coli中,TetR會與目標基因的tetracycline(tet) 操縱子結合,阻止基因進行轉錄作用。在加入誘導劑tetracycline之後,誘導劑會與TetR結合,造成抑制子的構形改變,使TetR離開操縱子而使目標基因得以表現。因此,本實驗希望(1)利用Gateway位點特異性重組系統來建構一快速且簡便的反轉錄病毒載體技術平台,再藉由病毒顆粒感染細胞,來評估此改良型之病毒載體在細胞內表現基因的效率 (2)探討不同promoter在慢病毒載體中對所攜帶之基因的表現情形。實驗的結果顯示,我們已經測試出最佳Gateway 位點特異性重組技術的反應條件,兩種不同來源的表達基因片段都成功置換於改良型之病毒載體且在細胞內表現基因的效率沒有明顯不同。當慢病毒載體攜帶置於miR155片段中所針對冷光酶所設計之miR155-shRNA時其對於冷光脢抑制效率高於攜帶miR30片段中所針對冷光酶所設計之miR30-shRNA之慢病毒載體。除此之外,我們成功的將適用於Gateway 位點特異性重組系統的重組片段置入含有不同啟動子的慢病毒載體中,並且發現在不同啟動子之慢病毒載體系統中,當WPRE存在時,含有UBC promoter之慢病毒載體表現冷光酶基因的情形最佳。總結以上,我們成功的建立快速簡便且具有高效率基因表達能力的慢病毒載體基因選殖技術,期待此快速簡便的改良型反轉錄病毒載體技術平台對未來基因治療的應用上有所幫助與貢獻。
Lentiviruses are the type of retrovirus that can infect both dividing and nondividing cells. Lentiviruses can be used to provide highly effective gene transfer as well as gene therapy. Despite of success, traditional gene cloning for viral vector included choosing suitable restriction enzyme and confirming the gene orientation remains a major obstacle. Recently, scientists developed a new gene cloning technique called Gateway Recombination System. This system allowed transfer of DNA fragments by specific recombination site between different cloning vectors without restriction endonucleases and ligase while maintaining the reading frame. Therefore, we employed Gateway Recombination System to establish a convenient lentiviral vector technical platform. For the application of gene therapy, choice of promoter affects the gene expression of the vector. There are many kinds of RNA polymerase II promoter including CMV, UBC, and inducible CMV-TO promoter. These promoters have their own characteristic. CMV promoter is a viral promoter and is often thought of as a constitutively active unregulated promoter. Human ubiquitin C (UBC) promoter is a endogenous promoter and highly conserved in all eukaryotic cell. So it can constitutively express gene in mammalian cell. And CMV-TO promoter is a promoter based on tetracycline repressor (TetR) inducible system. To accomplish our goal, we studied: (1) to characterize gene expression efficiency in the lentiviral vector with gateway recombination, (2) to investigate gene expression efficiency in the lentiviral vector with different promoter. Our results shown: We have already found the best reaction condition of gateway recombination and successfully exchanged gene sequence into improved viral vector. In microRNA-based RNA interference miR-shRNA system, we found that compared with viral vector containing miR30 shRNA targeted luciferase gene in miR30 cassette, the viral vector with miR155 shRNA in miR155 cassette has better inhibition efficiency of luciferase activity. Furthermore, we have successfully adopted the Gateway Recombination System for efficient route for subclone different promoters into lentiviral systems. Moreover, we also found that the lentiviral vectors containing both human ubiquitin C promoter and WPRE fragment has higher gene expression efficiency when compared with other promoters. In conclusion, we have successfully established an extremely fast and convenient viral vector cloning technique and higher gene expression efficiency in lentiviral systems. This system would provide us an opportunity to improve usefulness of lentiviral vector for gene therapy in near future.
目 錄
指導教授推薦書........................................i
口試委員會審定書......................................ii
授權書...............................................iii
誌謝.................................................iv
中文摘要..............................................v
英文摘要..............................................vi
縮寫表................................................vii
第一章 背景介紹........................................1
1. 病毒載體............................................1
2. 啟動子..............................................6
3. 報導基因系統.........................................8
4. RNA干擾現象.........................................11
5. 表達shRNA之啟動子....................................14
6. Gateway位點特異基因重組的系統.........................15
第二章 研究動機與目的...................................17
第三章 材料和方法.......................................18
第四章 實驗結果.........................................39
1. 建立可利用Gateway位點特異性重組系統的病毒載體............39
2. 建立可以表達microRNA30以及microRNA155 based shRNA的載體平台.....................................................40
3. 在病毒載體中比較microRNA30以及microRNA155 based
shRNA基因抑制的效果......................................42
4. 利用Gateway位點特異性重組系統比較不同promoter對於
在反轉錄病毒載體系統中基因表達的效率........................44
5. 測試帶有gateway位點特異性重組系統之病毒載體表達基因
效率之能力...............................................47
第五章 討論.............................................49
參考文獻.................................................58
圖表....................................................65

圖表目錄
圖一、Gateway位點特異性重組系統示意圖.......................65
圖二、pLenti6/V5-DEST、pLenti6/CMVTO/V5-DEST、pLenti6/Ubc/ V5-DEST之載體示意圖.......................................66
圖三、pLenti-V5-DEST-WPRE、pLenti/CMVTO/V5-DEST-WPRE、pLenti/Ubc/V5-DEST-WPRE之載體示意圖.......................67
圖四、pGiNWF-V5-DEST與c-FUW/CMV/V5-DEST E之載體示意圖......68
圖五、BLOCK-iT Pol Ⅱ miR RNAi Expression Vector Kits (miR155)中primer annealing示意圖..........................69
圖六、pcDNA6 BLOCK-iT Pol Ⅱ miR RNAi Expression Vector
Kits (miR30) 中利用template primer以及帶有XhoⅠ及EcoRⅠ的primer進行PCR之示意圖.....................................70
圖七、建構可表達microRNA之載體示意圖........................71
圖八、以HEK293FT 細胞測試pcDNA6.2-GW-MRed-miR 系列載體
抑制EGFP的效果............................................72
圖九、比較pcDNA6.2-GW-MRed-miR 系列載體抑制冷光酶(Luciferase)
的效果....................................................73
圖十、在pGiNWF病毒載體中比較不同microRNA backbone抑制冷光
酶(Luciferase)的效果......................................74
圖十一、將病毒定量後,比較在pGiNWF病毒載體中比較不同
microRNA backbone抑制冷光酶(Luciferase)的效果..............75
圖十二、比較含有不同promoter的Lentiviral vector包裹成病毒顆粒後
,基因表達的效率...........................................76
圖十三、比較在WPRE片段存在下含有不同promoter的Lentiviral
vector基因表達的效率......................................78
圖十四、將病毒定量後,比較在WPRE片段存在下含有不同promoter
的Lentiviral vector基因表達的效率.........................80
圖十五、測試帶有gateway位點特異性重組系統之病毒載體表達基因
效率之能力...............................................81
表一、針對冷光酶所設計之shRNA 序列 ........................83
表二、本論文所建構的引子序列...............................84
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