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研究生:羅思成
研究生(外文):Sih-Cheng Luo
論文名稱:α-乳白蛋白/第一型類胰島素生長因子基因轉殖乳山羊之產製與分析
論文名稱(外文):Generation and Analysis of αLA-IGF-1 Transgenic Dairy Goat
指導教授:蘇弘毅蘇弘毅引用關係
指導教授(外文):Hung-Yi Su
學位類別:碩士
校院名稱:國立屏東科技大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
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:基因轉殖乳山羊顯微注射
外文關鍵詞:IGF-1transgenic goatmicroinjection
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第一型類胰島素生長因子(insulin-like growth factor 1;IGF-1)其蛋白質功能與胰島素相似,扮演共同調節與媒介生長激素(growth hormone;GH)的內分泌及自體/旁分泌之功能,在哺乳動物出生後個體的生長,以及乳腺組織生長、發育、形態和泌乳量的表現上亦為重要角色和指標,並且利用IGF-1亦可治療人類之生長不全與糖尿病之問題。在先前的研究中已成功將牛α-乳白蛋白(α-lactalbumin;α-LA)啟動子及IGF-1之cDNA基因序列構築形成之αLA-IGF-1轉殖基因注入小鼠胚原核中,且已產製該基因轉殖小鼠。因此在本研究中再嘗試將該基因注入乳山羊胚之原核中,期望產製攜帶有該外源基因之轉基因乳山羊,並探討影響此產製過程中的各種因素與其可行性。在乳山羊原核胚收集實驗中,藉由重複使用及不同季節超級排卵羊隻,搭配不同胚體內人工受精策略之處理,用以探討對於排卵數與受精率之影響;使用未經超排與重複超排處理之羊隻在排卵數分別為13.27個/頭(n=22)與11.65個/頭(n=20),於繁殖季節與非繁殖季節超排處理之排卵數分別為12.03個/頭(n=31)與13.82個/頭(n=11);在受精策略上使用自然配種、新鮮精液、冷凍精液搭配自然配種與新鮮精液搭配自然配種之方式,其受精率(原核胚數總合/回收卵數總合)分別為5﹪(4/74)、25﹪(67/269)、34﹪(23/70)與29﹪(22/75);結果顯示以冷凍精液搭配自然配種之體內人工受精策略為最有效率之原核胚收集方式,而超排的羊隻與季節之不同並無顯著影響其排卵效果。在αLA-IGF-1之基因轉殖試驗中,合計目前完成41個胚之基因注射,經移置12頭受胚母羊之輸卵管後,僅3頭受胚羊順利懷孕分娩,獲得4頭公仔羊與2頭母仔羊之出生;另有1頭受胚羊流產,獲得3頭死胎仔羊。經剪取仔羊耳部組織分別抽取DNA進行PCR分析,並無攜帶轉殖之基因。截至目前仍有3頭懷孕之受胚羊於待產中。
第1章 前言
第2章 文獻檢討
2.1研究基因轉殖乳山羊之源起與概況
2.2基因轉殖乳山羊超級排卵與發情同期化
2.2.1乳山羊季節性之繁殖機制
2.2.2超級排卵與同期化發情之應用
2.2.3應用上常見之方法
2.3第一型類胰島素生長因子
2.3.1第一型類胰島素生長因子之結構與基因
2.3.2第一型類胰島素生長因子之生成與功能
2.3.3類胰島素生長因子與結合蛋白間之關係
2.3.4第一型類胰島素生長因子與生長激素之關係
2.3.5第一型類胰島素生長因子與乳腺發育和乳汁生產之關係
2.4產製轉基因動物之方法
2.4.1病毒轉染法
2.4.2精子載體法
2.4.3胚幹細胞法
2.4.4體細胞核移置法
2.4.5原核顯微注射法
2.5基因轉殖家畜於生物醫學與農畜上之應用
2.5.1增強抵抗疾病能力
2.5.2人體異種器官移置
2.5.3提升家畜生產性能
2.5.3.1改進生長或體組成及飼料利用效率
2.5.3.2改良乳汁成分或品質
2.5.3.3改善羊毛生產量
2.5.4作為生物反應器
2.5.4.1家畜產製生物反應器之系統
2.5.4.2乳蛋白基因與啟動子
第3章 材料與方法
3.1轉殖基因表現載體之構築策略
3.1.1 αLA-IGF-1基因構築
3.1.2質體DNA之製備
3.1.3質體DNA片段之截切與回收
3.1.4轉殖基因之純化
3.2試驗動物之飼養管理與超級排卵策略
3.2.1試驗用乳山羊
3.2.2試驗用乳山羊之超級排卵與同期化發情處理方法
3.3試驗用乳山羊之體內人工受精處理策略
3.4腹中線沖胚手術
3.5山羊原核胚之顯微基因注射
3.6基因轉殖動物之分析
3.7統計分析
第4章 結果
4.1 aLA-IGF-1表現載體之構築
4.2基因轉殖乳山羊之產製與分析
4.2.1乳山羊原核胚收集之結果
4.2.2不同受精策略之結果
4.2.3 aLA-IGF-1基因轉殖乳山羊之產製與分析
第5章 討論
5.1 供胚乳山羊超級排卵之效果
5.2 供胚乳山羊受精之效果
5.3 供胚乳山羊個體差異之影響
5.4 顯微注射之外源基因嵌插與表現效果
5.4.1 外源基因型態之影響
5.4.2 注射前處理之影響
5.4.3 外源基因序列之影響
5.4.4 外源基因嵌插位置之影響
第6章 結論
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