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研究生:方誠傑
研究生(外文):Cheng-Chieh Fang
論文名稱:以RNA干擾作YM1基因調控之研究
論文名稱(外文):Conditional gene silencing of Ym1 by RNA interference
指導教授:張南驥
指導教授(外文):Nan-Chi Chang
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:微生物及免疫學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:中文
論文頁數:74
中文關鍵詞:RNA干擾陽明一號
外文關鍵詞:RNA interferenceYM1siRNAalternative activationmacrophage
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YM1是老鼠感染旋毛蟲腹腔巨噬細胞所分泌的蛋白質。YM1是單鏈的蛋白質,分子量約45KDa,在等電點5.7時具有很強的結晶能力。YM1是YM家族七個成員之一位於老鼠第三號染色體上。YM1是一種類凝集素(lectin-like)的蛋白質,可以鍵結具胺基(amine group)的多醣類,在生理上硫酸乙醯肝素(heparan sulfate)似為其配基。在胚胎發育時期,YM1的表現伴隨造血組織位置遷移而改變。在不同的發炎實驗模組中也發現發炎反應會誘導YM1表現,但其功能目前仍然未知。RNA干擾(RNA interference)是個功效強大的技術,可在mRNA的層次專一性地降低蛋白質表現,適用於YM1功能之研究。配合四環黴素誘導系統(tetracycline regulatory system)的調控,可以在不同時期調控RNA干擾的表現,增加RNA干擾之效用。
整合相關文獻提供之規則預測出三個干擾YM1表現的目標序列(target sequence)來壓抑YM1在細胞中的表現,命名為YM1-114、YM1-710和YM1-1040。把這三個目標序列搭配一個序列當成環(loop)設計成可轉錄成髮夾狀RNA的模式,分別建構在具有H1啟動子(promoter)的載體(pSuper-basic)上,之後把表現YM1之載體和RNA干擾之載體依1:2、1:4和1:8的莫耳比例共同轉染到293T細胞株中測試,從西方點墨法的資料分析, YM1-114降低YM1蛋白質表現至27%、20%、18%,YM1-710降低至45%、49%,54%,YM1-1040降低至77%、56%、40%,因此根據以上資料得知YM1-114是個很有淺力的目標序列。而配合四環黴素啟動系統,加入0µg/ml到100µg/ml之間不同劑量的去氧弳脛四環素(doxycycline)後,從西方點墨法的資料分析得知YM1蛋白質的表現隨著去氧弳脛四環素增加逐漸減少,分別降至81%、136%、87%、33%、20%、14%、14%,確定這樣的策略的確可以在培養的細胞中調控RNA干擾抑制YM1蛋白質之表現。
本論文確定了YM1-114這個目標序列能有效地壓抑YM1蛋白質的表現,有助於未來RNA干擾YM1表現之基因轉殖老鼠建立,然而以載體方式操作RNA干擾在老鼠原表現YM1蛋白質的細胞中測試是必要的,特別是要考慮到啟動子效率。最後,以基因工程方法結合藥物調控系統與RNA干擾技術,建立活體動物的實驗模組是本實驗最終的目的。
Abstract
YM1 is a secretory protein produced by peritoneal macrophages of mice infected with Trichinella spiralis. It is a single-chain polypeptide with molecular weight of 45KDa and tends to crystallize at pI of 5.7. YM1 is a member of YMx family consists of 7 homologous genes clustered on mouse chromosome 3. YM1 is a lectin-like protein with binding specificity to an amine group of polysaccharides. Heparan sulfate is likely the physiological ligand of YM1. The temporal and spacial expression of YM1 correlated to the sequential switch of hematopoietic sites during embryonic development, YM1 can be induced by inflammatory stimulus in many inflammatory models. However, the definitive function of YM1 in vivo is still unknown. RNA interference is a powerful technology to knock down gene expression by triggering a degradation of homologous mRNA. This is thus applicable to be used for analysing the functions of YM1. Under tetracycline regulatory controls, RNA interference can be expanded to broaden its adoptability by swiching up and down in particular period.
Utilizing empirical rules published for selection of siRNA segments, we predicted and used three target sequences to silence cellular expression of YM1, namely YM1-114, YM1-710 and YM1-1040. These three target sequences with known hairpin RNA loop are separately cloned into vector (pSuper-basic) driven by H1 promoter. Those RNAi vectors were used later with YM1-expression vector to cotransfect into 293T cell line with mole ratio of 1:2, 1:4 and 1:8. Western blot analyses indicate the target sequence YM1-114 reduces YM1 expression up to 27%、20%、18% on mole ratio of 1:2, 1:4 and 1:8, respectively. The target sequence YM1-710 reduces YM1 expression to 45%、49%,54% and YM1-1040 reduce YM1 expression to 77%、56%、40%, respectively. The target sequence YM1-114 is the best candidate to silence YM1 expression according to above data. Tet-on based RNAi-YM1-114 once subjected to different doses of doxycycline, the western blot data indicate YM1 expression can be reduced up to 81%、136%、87%、33%、20%、14%、14% with increase of concentration of doxycycline ranging from 0µg/ml to 100µg/ml. These data confirm that the strategy of conditional RNA interference in cell culture system is feasible.
In this thesis, we idendify the target YM1-114 as effective to knock down YM1 expression. This will further facilitate the setup of RNAi-YM1 transgenic mice. However, testing the efficiency of vector-based RNAi in primary cells expressing YM1 is required, particularly adverting different promoters effect. Finally, conditionally swiching up RNAi in drug-controled system in genetically engineered live animal model systems will be our eventual goal.
目錄
Abstract 1
摘要 3
緒論 5
材料與方法 11
RNA干擾技術設計(Design RNA interference) 11
勝任細菌之製備(Competent cell) 12
質體轉化作用(Transformation) 13
菌落雜合反應(Colony hybridization) 13
小量抽取質體DNA(Viogene mini-prep)及菌種保存 15
大量抽取質體DNA(QIAGEN Plasmid Midi Kits) 15
建構RNA干擾抑制YM1蛋白質表現的質體(Construct pSuper-YM1) 17
建構調控RNA干擾抑制YM1蛋白質表現之質體(Construct Inducible pSuper-YM1) 18
建構調控RNA干擾抑制YM1蛋白質表現之慢病毒質體(Construct pLVTHM-YM1) 19
建構表現tTR/KRAB蛋白質的質體(Construct ptTR/KRAB-Red-Neo) 20
建構表現YM1蛋白質的質體(Construct pcDNA3-YM1) 21
細胞培養(Cell culture) 22
細胞的繼代培養(Subculture) 22
細胞的冷凍保存(Conservation) 22
冷凍細胞的解凍(Defreeze) 23
轉染作用(Transfection) 23
流式細胞儀(Flow cytometry) 24
螢光顯微鏡(Fluorescence microscopy) 25
西方點墨法(Western blot) 25
即時螢光定量聚合酵素連鎖反應(Real-time PCR) 27
結果與分析 31
RNA干擾技術之設計 31
實驗建構的質體之正確性 31
293T細胞株轉染之效率 32
轉染pcDNA3-YM1的293T細胞株YM1蛋白質之表現 33
RNA干擾降低YM1表現之分析 34
轉染ptTR/KRAB-Red-Neo的293T細胞株tTR/KRAB融合蛋白質之表現 35
四環黴素誘導系統調控RNAi干擾YM1蛋白質之表現 36
討論 38
參考文獻 46
圖表………………………………………………………………………………….52
參考文獻

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