Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/22590
DC FieldValueLanguage
dc.contributor蘇鴻麟zh_TW
dc.contributor王繼廣zh_TW
dc.contributor.advisor鄭旭辰zh_TW
dc.contributor.author毛敏驊zh_TW
dc.contributor.authorMao, Ming-Huaen_US
dc.contributor.other中興大學zh_TW
dc.date2009zh_TW
dc.date.accessioned2014-06-06T07:18:18Z-
dc.date.available2014-06-06T07:18:18Z-
dc.identifierU0005-2901200809240800zh_TW
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dc.identifier.urihttp://hdl.handle.net/11455/22590-
dc.description.abstract當細胞受到外來刺激時,會產生許多機制去做不同的調節,通常會需要合成新的mRNA以及蛋白質。在mRNA的層級,對於調節細胞的反應情形可以由Real-time PCR(即時連鎖聚合酶反應)來得知訊息。然而這些結果卻無法應用在轉譯前或轉譯的蛋白質上面。為了要能得知細胞如何去反應外來訊息時,而且必須能在蛋白質上得到正確地以及並可重複性地定量蛋白質的量與其活性。但是現今發現一般短暫的轉染作用並沒有辦法能夠去精準分析測出轉錄因子的活性。因此我們想要在in vivo或是在in vitro中發展出一個平台利用結合Cre/loxP系統與雙冷光酵素分析系統作為分析定量出轉錄因子的活性或數目。為了將細胞的變化值減到最小,我們設計了帶有firefly luciferase reporters穩定細胞株的建立,並且利用fusion protein(同時帶有Renilla luciferase與轉錄因子構築融合蛋白(fusion protein))去計算出單獨轉錄因子的數量。為了要能縮短建立多種穩定細胞株的時間,因此我們使用Cre/loxP系統能有效做目標序列的轉換。我們分別利用DF-1與3T3這兩株細胞株,去建立帶有reporters的穩定細胞株,期望其細胞株中帶有的lox sites能夠快速地將目標序列進行置換。更進一步地,希望這個系統能夠被應用於不同的蛋白質在轉殖動物中的功能。zh_TW
dc.description.abstractTo react to extracellular stimuli, cells employ various mechanisms to regulate different cellular responses, which usually require synthesis of novel mRNAs and proteins. Real-time PCR has provided valuable quantitative information on the regulation of cellular responses at mRNA level; however those results are not applicable to proteins that have translational or post-translational control. To obtain accurate and reproducible quantitative information on how cells respond to extracellular signals at protein level, we found the conventional transient transfection strategy that can not precisely measure transcription factors activity is not sufficient. Therefore, we intend to develop a platform that combines Cre/loxP system and dual luciferase reporter system for quantitative analysis of transcription factors activity in vitro and in vivo. To minimize cellular variations, we generated stable cell lines for firefly luciferase reporters, and used Renilla luciferase-fused transcription factor to measure the quantity of individual transcription factor. Also to facilitate the time-consuming processes of generating multiple stable cell lines, we used Cre/loxP system for efficient target sequence swapping. To demonstrate the practical applications of this strategy, we generated a collection of reporter cell lines from DF-1 and NIH/3T3 cells containing lox sites for rapid exchange of target DNA sequences. Further, this system can be applied to study the function of different proteins in transgenic animals.en_US
dc.description.tableofcontents中文摘要…………………....................................................................................i 英文摘要...............................................................................................................ii 目錄……………………………………………………………….…………….iii 圖表目錄……………………………………………………….……………….iv 名詞縮寫表….…………………………………………..……….…………….viii 緒論 一、 目的與策略….……………………………………………………... 1 二、 報導基因分析….………………………………………....................2 三、 Cre/loxP 系統….………………………………………....................4 四、 細胞株篩選….……………………………………………………....6 五、 GLi1-GliBS….…………………………………………...........…….7 六、 Gal4-UAS….……………………………...………………………...9 材料與方法 一、 細胞株….…………………………………………………………..10 二、 細胞培養….………………………………………………………..10 三、 質體的製備….……………………………………………………..11 四、 細胞轉染作用….…………………………………………………..16 五、 建立穩定細胞株….………………………………………………..16 六、 蛋白質定量分析….………………………………………………..17 七、 冷光報導基因分析….……………………………………………..17 八、 細胞株基因型(Genotype analysis)分析: 聚合酶連鎖反應(PCR)分析…………………………………….18 結果 一、 DNA 重組質體之設計與構築……………………………………20 二、 建立『重組起始細胞株』………………………………………. 21 三、 在細胞內進行DNA 重組…………………………………………21 四、 篩選DNA 重組細胞株……………………………………………21 五、 各細胞株之活性分析…………………………………..................22 六、 轉錄子活性之量化分析………………………………..................22 七、 聚合酶連鎖反應(PCR)分析……………………………………………24 討論……………………………………………………………………………..25 參考文獻…………………………………………………………………..……27 實驗結果表圖…………………………………………………………………..33 附圖……………………………………………………………………………..65 圖表目錄 表一、利用報導基因分析系統偵測在DF-1 中所建立重始穩定細胞株(TK) 之活性。 ………………………………………………………………..33 表二、利用報導基因分析系統偵測在3T3 中所建立重始穩定細胞株(TK) 之活性。………………………………………………………………...34 表三、PCR primers…………………………………………………………....35 表四、比較TK與GliBS 類似的序列………………………………….…...…..36 表五、比較TK與UAS類似的序列…………………………….…………..….36 圖1. pF5………………………………………………………………………37 1.1 primers 的設計以及定序的結果 1.2 質體構築 1.3 利用PvuⅡ和 SpeI 切割pF5 之瓊酯膠圖 1.4 pF5 酵素位置圖 圖2. pGL-F5………………………………………………………… ………38 2.1 質體構築 2.2 利用HindⅢ 切割pGL-F5 之瓊酯膠圖 2.3 利用BglⅡ和 SalI 切割pGL-F5 之瓊酯膠圖 圖3. pGL-F5-TK……………………………………………………………..39 3.1 質體構築 3.2 利用XbaI 切割pGL-F5-TK 之瓊酯膠圖 3.3 利用XbaI 和 SpeI 切割pGL-F5-TK 之瓊酯膠圖 圖4. pF5-GliBS……………………………………………………… ……...39 4.1 質體構築 4.2 利用XhoI 和HindⅢ切割pF5-GliBS 之瓊酯膠圖 圖5. pF5-UAS………………………………………………………………..40 5.1 質體構築 5.2 利用PvuⅡ切割pF5-UAS 之瓊酯膠圖 圖6. pGL-F5-UAS…………………………………………………………...40 6.1 質體構築 6.2 利用XhoI 和XbaI 切割pGL-F5-UAS 之瓊酯膠圖 圖7. pcDNA-Cre……………………………………………………………..41 7.1 質體構築 7.2 利用ClaI 和 MluI 切割pcDNA-Cre 之瓊酯膠圖 圖8. pTOPO-GAL4………………………………………………………….41 8.1 質體構築 8.2 利用ClaI 和NcoI 切割pTOPO-GAL4 之瓊酯膠圖 圖9. pL-GAL4…………………………………………………………..…....42 9.1 質體構築 9.2 利用EcoRI 和SalI 切割pL-GAL4 之瓊酯膠圖 圖10. pTOPO-GAL4-PCR、 pTOPO-SVRL-PCR…………………………42 10.1 pTOPO-GAL4-PCR 質體構築 10.2 利用HindⅢ切割pTOPO-GAL4-PCR 之瓊酯膠圖 10.3 pTOPO-SVRL-PCR 質體構築 10.4 利用HindⅢ切割pTOPO-SVRL-PCR 之瓊酯膠圖 圖11. pSVRL-GAL4………………………………………………………...…43 11.1 質體構築 11.2 利用BamHI 和BsrGI 切割pSVRL-GAL4 之瓊酯膠圖 11.3 pSVRL-GAL4 定序結果之比對 圖12. 利用報導基因分析系統測出在不同細胞株中,重組起始細胞株 (TK)與置換重組細胞株(GliBS、UAS)活性變化的狀態。………… 45 圖13. 將DF-1 細胞株進行不同質體短暫轉染,並利用報導基因分析系統 偵測冷光酵素活性。……………………………………………………46 13.1 短暫轉染pSV-RL、pGL-F5-TK、pSV-GLiRL、pGL-GliBS 13.2 短暫轉染pSV-RL、pGL-F5-TK、pSVRL-GAL4、pGL-F5-UAS 圖14. 重組起始細胞株DF-1 E11(TK)與置換重組細胞株(E11-GliBS、 E11-UAS),進行不同質體短暫轉染,並利用報導基因分析偵測冷 光酵素活性。…………………………………………………….. …..48 14.1 短暫轉染pSV-RL、pSV-GLiRL 14.2 短暫轉染pSV-RL、pSVRL-GAL4 圖15. 重組起始細胞株DF-1 F1(TK)與置換重組細胞株(F1-GliBS、 F1-UAS),進行不同質體短暫轉染,並利用報導基因分析偵測冷 光酵素活性。…………………………………………………….........50 15.1 短暫轉染pSV-RL、pSV-GLiRL 15.2 短暫轉染pSV-RL、pSVRL-GAL4 圖16. 重組起始細胞株DF-1 B7(TK)與置換重組細胞株(B7-GliBS、 B7-UAS),進行不同質體短暫轉染,並利用報導基因分析偵測冷 光酵素活性。………………………………………………………….52 16.1 短暫轉染pSV-RL、pSV-GLiRL 16.2 短暫轉染pSV-RL、pSVRL-GAL4 圖17. 將3T3 細胞株進行不同質體短暫轉染,並利用報導基因分析系統 偵測冷光酵素活性。……………………………………………………54 17.1 短暫轉染pSV-RL、pGL-F5-TK、pSV-GLiRL、pGL-GliBS 17.2 短暫轉染pSV-RL、pGL-F5-TK、pSVRL-GAL4、pGL-F5-UAS 圖18. 重組起始細胞株3T3 C3(TK)與置換重組細胞株(C3-GliBS、 C3-UAS),進行不同質體短暫轉染,並利用報導基因分析偵測冷 光酵素活性。…………………………………………………………..56 18.1 短暫轉染pSV-RL、pSV-GLiRL 18.2 短暫轉染pSV-RL、pSVRL-GAL4 圖19. 重組起始細胞株3T3 H5(TK)與置換重組細胞株(H5-GliBS、 H5-UAS),進行不同質體短暫轉染,並利用報導基因分析偵測冷 光酵素活性。………………………………………………………….58 19.1 短暫轉染pSV-RL、pSV-GLiRL 19.2 短暫轉染pSV-RL、pSVRL-GAL4 圖20. 重組起始細胞株3T3 D4(TK)與置換重組細胞株(D4-GliBS、 D4-UAS),進行不同質體短暫轉染,並利用報導基因分析偵測冷 光酵素活性。…………………………………………………………..60 20.1 短暫轉染pSV-RL、pSV-GLiRL 20.2 短暫轉染pSV-RL、pSVRL-GAL4 圖21. 短暫轉染與穩定細胞株(GliBS 或UAS)加入effector 之後,比較轉 錄因子活性增加的比例以及其穩定性。………………………………62 21.1 短暫轉染與穩定細胞株(GliBS)加入pSV-Gli1RL 之比較 21.2 短暫轉染與穩定細胞株(UAS)加入pSVRL-GAL4 之比較 圖22. 利用PCR 確認基因體上TK、Luc、GliBS、UAS 之存在… ……….63 22.1 DF-1 細胞株 22.2 3T3 細胞株 附圖一、報導基因發光原理…………………………………………………..65 附圖二、Cre 重組酶結晶之結構………….……………………………… …66 附圖三、loxP 的序列組成以及Cre 重組酶結合在loxP 序列上的位置..….66 附圖四、特定位置重組作用之示意圖………………………………………...66 附圖五、Cre 重組酶所造成的插入、反轉、刪除以及易位之示意圖………67 附圖六、正常與產生突變的loxP 其序列…………………………………….67 附圖七、正常與產生突變的loxP 在E. coli 中會進行重組的機率…………67 附圖八、pGL3-promoter 質體建構………………………………………………68 附圖九、pPNT 質體建構……………………………………………………......68 附圖十、pUAST 質體建構……………………………….………………..........69 附圖十一、pPTGAL 質體建構……………………………………………………69zh_TW
dc.language.isoen_USzh_TW
dc.publisher生命科學系所zh_TW
dc.relation.urihttp://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-2901200809240800en_US
dc.subject穩定細胞株zh_TW
dc.subjectcreen_US
dc.title利用一個新技術去快速建立DF-1及NIH 3T3穩定細胞株zh_TW
dc.titleA novel strategy of establishing an in vitro assay system in DF-1 and NIH3T3 cell linesen_US
dc.typeThesis and Dissertationzh_TW
item.cerifentitytypePublications-
item.grantfulltextnone-
item.languageiso639-1en_US-
item.fulltextno fulltext-
item.openairecristypehttp://purl.org/coar/resource_type/c_18cf-
item.openairetypeThesis and Dissertation-
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