臺灣博碩士論文加值系統

本研究旨在將構築有牛α-乳白蛋白基因(α-lactalbumin ; αLA)調節序列及豬乳鐵蛋白(porcine lactoferrin, pLF)cDNA或人類凝血第九因子(human factor IX, hFIX)cDNA序列之轉殖基因，長度分別為4.3kb及4.9 kb，應用顯微注射技術注入小鼠與豬等早期胚之原核中，再以胚移置技術分別移置於受胚小鼠與受胚豬之生殖道內，冀能產製攜有前述轉殖基因，且能在乳腺表現高水平基因產物之基因轉殖小鼠與基因轉殖豬，俾能從而改善仔豬下痢及貧血問題，並可藉由豬之乳腺量產具生物活性之高價位的人類凝血第九因子。
試驗結果證明在αLA-pLF之基因轉殖試驗中，合計完成682個小鼠胚及288個豬胚之基因注射，經分別移置於受胚母小鼠及受胚母豬之輸卵管後，各獲得81隻仔小鼠及47頭仔豬之出生。經剪取尾部組織抽取基因組DNA，進行PCR、Slot-blot 及Southern-blot等分析結果，證實其中9(3♀/6♂)隻仔小鼠及5(0♀/5♂)頭仔豬分別係為基因轉殖成功者；其中除2(♀)隻小鼠不孕外，另7(1♀/6♂)隻均已明確證實可將轉殖基因傳承於後代，進一步採集基因轉殖母小鼠之乳汁及乳腺組織，經西方吸漬分析法、酵素免疫分析法及組織切片免疫化學染色法分析結果更證明確有表現豬乳鐵蛋白之事實，最高表現量達100μg/ml。另在獲得之5頭基因轉殖豬中，僅1頭存活迄性成熟階段，經配種測試結果證實，其轉殖基因亦具有在性腺傳承之事實。取其分娩後之乳汁，經西方吸漬分析結果證實，自泌乳期第1天迄第28天均可表現外源性之豬乳鐵蛋白。此外在αLA-hFIX基因轉殖試驗中，合計完成699個小鼠胚及212個豬胚之基因注射，經分別移置於受胚母小鼠及受胚母豬之輸卵管後，合計獲得 14隻基因轉殖小鼠與2頭基因轉殖豬。其中9隻雄性之轉殖基因係可在性腺中被傳承者。針對此基因轉殖小鼠各家族之母小鼠進行配種，于分娩後所收集之乳汁樣品，進行西方吸漬分析結果，證實其乳汁中含有hFIX之濃度平均約為45.64±16.32μg/ml，凝血活性測試之結果，亦證實此等hFIX之活性約為正常人血漿之9.15±0.61%。前述2頭(1♀/1♂)基因轉殖豬係同時注入αLA-pLF及αLA-hFIX兩個轉殖基因者；在PCR 及Southern-blot等分析結果，亦證明該2頭基因轉殖豬確係同時攜有二個轉殖基因者。進一步配種測試結果，不僅證實該二轉殖基因在性腺順利被傳承，且源自此等雌性基因轉殖豬所收集之乳汁，經西方吸漬法分析結果證明其含有之hFIX，平均高達408.34±55.00 μg/ml；此一高水平含量並有隨泌乳期之進展而升高之現象。此外，凝血活性測試之結果，更證實此等hFIX之活性約為正常人血漿之14.18±2.45%。

The purpose of this study was to generate transgenic mice and pigs by microinjection of the αLA-pLF and /or αLA-hFIX transgene contructs, containing the regulatory sequences of bovine α-lactoalbumin (αLA) and the structure sequences of cDNA from porcine lactoferrin (pLF) and/ or the human factor IX, into the pronucleus of newly fertilized mouse and porcine eggs, respectively. The transgene were chosen for the production of hFIX as a pharmaceutical product in the milk of transgenic animals and as a method to protect piglets from diarrheal diseases and anemia.
Transfer of 682 mouse embryos, after microinjection of the αLA-pLF transgene, into the fallopian tubes of 18 recipient mice had resulted in 14 mice becoming pregnant, 81 newborn mice were obtained from the embryo-transferred recipients. Nine (3♀/6♂) mice when subjected to PCR and Southern-blot analyses were confirmed to carry integrated transgene. Offsprings of seven (1♀/6♂) out of 9 transgenic founders, mated with normal ICR mice had their tail DNA extracted and the transgene was detected. The transgene found was to be stably integrated in the germ line. Screening by Western-blot analysis confirmed that expression of αLA-pLF transgene with pLF concentrations around 100 μg/ml were found in the milk obtained from five families of the transgenic mice produced.
Similarly, a total of 699 embryos, after injection of αLA-hFIX gene, were transferred into 18 recipient mice which resulted in 14 (77.8%) becoming pregnant and 89 pups were obtained at parturition. Fourteen out of 89 (15.7%) pups were confirmed to be transgenic when the genomic DNA from their muscle tissues had been subjected to PCR and Southern analysis. Of the 14 transgenic mice, 3 died soon after birth. Eleven were sexually matured. The only two transgenic females are found to be infertile after mating with several fertile males. All of 9 αLA-hFIX male transgenic founder mice are fertile and the transgene has been found to be germline-transmitted to between 7.1 and 73.3% of the F1 progenies obtained. Western-blot analysis of milk samples obtained from two of the F1 transgenic families has further shown that the milk contains hFIX in in a concentration ranged of 46g/ml.
Attempts were then made to generate transgenic piglets carrying the αLA-pLF and αLA-hFIX sequence(s). In these experiments a mixture of the αLA-hFIX DNA and αLA-pLF gene constructs was used in microinjection. A total of 500 pig embryos after gene injection were transferred into 19 recipient gilts resulting in 11 becoming pregnant and 58 piglets were obtained. Seven (1♀/6♂) of the newborn piglets were confirmed to be transgenics, according the results from PCR and Southern-blot analysis. While 4 male transgenic piglets died soon after birth, three (1♀/2♂) of the newborn piglets were confirmed as transgenic. Foreign gene of transgenic founder pigs was transmitted to F1 generation progenies with transmission rate ranged between 66.7% and 13.2%. Expression of hFIXcDNA and pLFcDNA in mammary gland was confirmed by Western-blot analysis of milk samples from the transgenic sow, and the concentration of hFIX contained in the milk ranged between 200∼500μg/ml, with a higher concentration of hFIX found in the milk of later lactation period. The hFIX clotting activity was assayed directly on milk collected from the transgenic sow, demonstrating that the hFIX synthesized by transgenic pig is active.

目 錄
頁次
目錄……………………………………………………………I
表次……………………………………………………………III
圖次……………………………………………………………IV
摘要………………………………………………………………1
緒言……………………………………………………………….2
第一章、文獻檢討……………………………………………….4
一、乳鐵蛋白…………………………………………4
二、人類凝血第九因子………………………………8
三、產製基因轉殖動物之方法………………………10
四、基因轉殖家畜之應用………………………………18
五、未來展望……………………………………… …23
第二章、試驗壹豬乳鐵蛋白cDNA在基因轉殖小鼠乳腺之表現…25
一、前言………………………………………………25
二、材料與方法………………………………………25
三、結果與討論……………………………………….31
第三章、試驗貳 豬乳鐵蛋白cDNA在基因轉殖豬乳腺之表現…36
一、前言………………………………………………36
二、材料與方法………………………………………37
三、結果與討論………………………………………41
第四章、試驗參人類凝血第九因子基因轉殖小鼠之產製及分析 46
一、前言…………………………………………………46
二、材料與方法………………………………………46
三、結果與討論………………………………………50
第五章、試驗肆 外源性豬乳鐵蛋白及人類凝血第九因子基因轉殖豬之產製及分析53
一、前言………………………………………………53
二、材料與方法………………………………………53
三、結果與討論………………………………………53
結論……………………………………………………………….58
參考文獻……………………………………………………………59
英文摘要……………………………………………………………74
表 次
頁次
表1. 偵測帶有LA-pLF和LA-hFIX基因之基因轉殖小鼠及豬時所使用之寡核酸引子配對……………………………………………
表2. 以基因顯微注射法產製LA-pLF基因轉殖小鼠………………..
表3. 豬乳鐵蛋白cDNA 在LA-pLF基因轉殖小鼠之嵌合及表現……
表4. 以基因顯微注射法產製LA-pLF基因轉殖豬…………………..
表5. 第一代LA-pLF基因轉殖豬之繁殖及性腺傳承表現…………...
表6. 含重組豬乳鐵蛋白豬乳經餵食哺乳期仔豬後對仔豬血球數目之影響…………………………………………………………………
表7. 哺乳期仔豬經沙門氏桿菌攻毒14天後對其腸道桿菌及總菌數之影響………………………………………………………………
表8. 乳鐵蛋白基因轉殖母豬與非基因轉殖母豬之繁殖性能…………
表 9. 以基因顯微注射法產製LA-hFIX基因轉殖小鼠………………
表10. αLA-hFIX基因轉殖小鼠與非基因轉殖小鼠配種後代之外源基因性腺傳承分析結果………………………………………….
表11. 基因轉殖小鼠乳汁中之重組人類凝血第九因子之凝血活性分析………………………………………………………………….
表12. 以基因顯微注射法產製LA-pLF及 LA-hFIX基因轉殖豬….
表13. LA-pLF及αLA-hFIX基因轉殖豬與非基因轉殖豬配種後代之外源基因性腺傳承分析結果………………………………………
表14. 基因轉殖豬乳汁中之組人類凝血第九因子之凝血活性分析……
圖 次
頁次
圖1. 凝血機制……………………………………………………………
圖2. LA-pLF轉殖基因之質體構築………………………………….
圖3. 以PCR方法進行LA-pLF基因轉殖小鼠之篩檢結果………….
圖4. 轉殖基因LA-pLF在基因轉殖小鼠基因組中之嵌插套數……..
圖5. LA-pLF基因轉殖小鼠之Southern-blot雜合分析(I)……………
圖6. LA-pLF基因轉殖小鼠之Southern-blot雜合分析(II)…………..
圖7. LA-pLF基因轉殖小鼠乳汁之西方吸漬法分析…………………
圖8. 泌乳第10天之 LA-pLF基因轉殖小鼠的組織切片免疫染色..
圖9. 第0 代LA- pLF基因轉殖豬之PCR分析及第0代LA-pLF基因轉殖豬之PCR雜合分析………………………………………..
圖10. LA-pLF基因轉殖豬之Southern-blot雜合分析……………….
圖11. 以PCR方法進行LA-pLF基因轉殖豬後代之篩檢結果………
圖12. 乳鐵蛋白基因轉殖豬(耳號 6-3)及其後代(耳號 6-3-1至6-3-19)之南方吸漬法分析……………………………………………….
圖13. LA-pLF基因轉殖(上圖)及非基因轉殖(下圖)豬乳汁之西方吸漬法分析……………………………………………………………
圖15. 不同泌乳期基因轉殖(064-11)及非基因轉殖(071-11)母豬乳樣之乳鐵蛋白酵素免疫分析……………………………………….
圖14. 不同泌乳期(第1, 14, 28天)基因轉殖(TGⅠ,Ⅱ,Ⅲ)及非基因轉殖(NTG)母豬乳樣之乳鐵蛋白酵素免疫分析…………………..
圖16. LA-hFIX轉殖基因之質體構築…………………………………
圖17. LA-FIX基因轉殖小鼠之南方吸漬分析圖(I)………………….
圖18. LA-FIX基因轉殖小鼠之南方吸漬分析圖(II)…………………
圖19. 以PCR方法進行LA-hFIX基因轉殖小鼠後裔之篩檢結果….
圖20. LA-FIX基因轉殖小鼠後裔之南方吸漬分析圖………………..
圖21. LA-hFIX 基因轉殖小鼠乳清之西方吸漬分析結果……………
圖22. LA-hFIX 基因轉殖小鼠乳清之西方吸漬分析結果……………
圖23. 源自第10天 LA-hFIX基因轉殖小鼠之乳腺組織切片免疫染色……………………………………………………………………
圖24. 以PCR方法進行LA-hFIX及LA-pLF基因轉殖仔豬之篩檢結果……………………………………………………………….
圖25. LA-PLF 和LA-hFIX 基因轉殖豬 (編號  8-3及 8-5) 及其子代(編號  8-3 -116及 8-5 -116 )基因組 DNA經HindIII限制酵素截切後進行南方雜合分析之結果……………………..
圖26. 豬乳鐵蛋白及人類凝血第九因子基因轉殖豬(No. 8-3)後代之PCR分析…………………………………………………………
圖27. 豬乳鐵蛋白及人類凝血第九因子基因轉殖豬 (No. 8-5) 後代之PCR分析………………………………………………………….
圖28. LA-hFIX 基因轉殖豬(編號  8-3) 及其子代(編號  8-3 -12 ; 8-3 -12及8-3-1416 )基因組 DNA經EcoRV 限制截切後進行南方雜合分析之結果……………………………………………
圖29. LA-hFIX 基因轉殖豬(No. 8-3)乳樣之西方吸漬分析(DAB)結果(I)…………………………………………………………………
圖30. LA-hFIX 基因轉殖豬(No.8-3)乳樣之西方吸漬分析(ECL)結果(II)……………………………………………………………
圖31. LA-hFIX及LA-pLF基因轉殖豬(No.8-5後裔)乳樣之西方吸漬分析結果…………………………………………………………
圖32. 泌乳期第28天豬乳腺之組織切片免疫染色圖………………….
圖33. 轉殖基因LA-pLF之序列………………………………………
圖34. 轉殖基因LA-hFIX之序列……………………………………..

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