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研究生:羅珮文
論文名稱:小鼠PGK-1啟動子的選殖與活性檢測
論文名稱(外文):Cloning and characterization of mouse PGK-1 promoter
指導教授:張文興
學位類別:碩士
校院名稱:國立嘉義大學
系所名稱:農業生物技術研究所
學門:農業科學學門
學類:農業技術學類
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
中文關鍵詞:轉殖基因動物北京鴨小鼠啟動子
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基因轉殖動物可以應用的範圍很廣,包括提高畜產的產量,次單位疫苗 (subunit vaccine) 的生產,以及醫用蛋白質的生產…等。而如何能使重組DNA質體送入細胞後表現量可以提高,是目前基因表現技術所面臨的重要挑戰;其中一個控制關鍵在於啟動子 (promoter)的表現強度。啟動子為基因表現所必需的DNA 序列,目前已有許多種類的啟動子可供表現蛋白質。由於在不同細胞中,表現性啟動子引導基因表現能力不完全相同,因此找到一種在動物體內可以穩定表現的啟動子顯得格外重要。本實驗是利用目前已知在哺乳類動物細胞內,表現度很好的啟動子小鼠PGK-1啟動子 (phosphoglycerate kinase-1 promoter)與報導基因 (reporter gene) 綠色螢光蛋白質 (EGFP , enhanced green fluorescence protein) 建構表現載體,並以精子-脂質體轉殖法 (sperm-liposome mediated method)轉殖到北京鴨,再以螢光顯微鏡以及PCR做檢測:以驗證小鼠PGK-1 promoter是否可在鴨的細胞內持續性表現,作為將來在製作基因轉殖禽類時,可以大量表現特定蛋白質的啟動子。
中文摘要 2
英文摘要 3
目錄 4
圖表目錄 8
縮寫檢索表 11
第一章序論 12
第一節 基因轉殖禽類的發展 12
1.基因轉殖動物目前的概況 12
2.基因轉殖禽類 13
3.基因轉殖禽類的方法 14
第二節 PGK的簡介及其啟動子的應用 18
1. PGK在細胞內的作用 18
2. PGK的構造及分類 19
3. PGK啟動子在轉殖基因動物的應用 20
第三節 綠螢光蛋白的簡介與其在基因轉殖動物的應用 22
1.綠螢光蛋白作用的原理 22
2.以綠螢光蛋白做為報導基因的應用 22
參考文獻 22
第二章 小鼠PGK-1啟動子的選殖 22
第一節 前言 22
第二節 材料與方法 22
1. 萃取基因組DNA(genomic DNA) 22
2. 放大PGK-1啟動子 22
3. 電泳分析(electrophesis) 22
4. 純化DNA 22
5. 選殖基因 22
6. 大腸桿菌勝任細胞的製備 22
7. 大腸桿菌的轉型作用 22
8. 萃取質體 22
9. 定序 22
第三節 結果 22
1. 以PCR大量製備小鼠的PGK-1啟動子序列片段 22
2. 將PCR放大的小鼠PGK-1啟動子與載體接合 22
3. 小鼠PGK-1啟動子定序的結果 22
附錄一 22
附錄二 22
參考文獻 22
第三章 建構小鼠PGK-1啟動子的表現載體與轉殖的基因在鴨細胞內的活性分析 22
第一節 前言 22
第二節 材料與方法 22
1.建構表現載體 22
2.製備線形載體 22
3.鴨隻的採精 22
4.轉染(transfect)鴨隻 22
5.鴨隻的人工授精 22
6.人工孵化鴨隻 22
7. PCR檢測 22
8. 綠螢光蛋白檢測 22
9. 定序 22
第三節 結果 22
1.構築表現載體 22
2.綠螢光蛋白的表現度 22
3.PCR檢測 22
4.表現載體序列的分析 22
參考文獻 22
第四章 結論 22
第一節 啟動子在轉殖基因動物的影響 22
第二節 小鼠PGK-1啟動子的選殖 22
第三節 建構表現載體與測試啟動子的活性 22
第四節 未來展望 22
參考文獻 22
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