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研究生:江潁彥
論文名稱:藉由KillerRed光敏蛋白誘發性腺退化以發展 基因轉殖斑馬魚不孕控制技術
論文名稱(外文):Infertility control of the genetically modified zebrafish by KillerRed-mediated gonadal dysgenesis
指導教授:胡紹揚 老師
學位類別:碩士
校院名稱:國立屏東科技大學
系所名稱:生物科技系所
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:2013
畢業學年度:101
語文別:中文
論文頁數:60
中文關鍵詞:斑馬魚光敏蛋白活性氧不孕控制
外文關鍵詞:ZebrafishKillerRedReactive oxygen speciesInfertility
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水生基因改造生物已廣泛研究於不同魚種以作為品質改良之應用,然而因全球擔心基改生物可能對生態造成基因流佈與原生種滅絕等問題,因此除基因轉殖觀賞魚外,水生基改生物產品仍窒礙難行。有鑑於此,建立不孕技術的開發為解決此課題的核心技術。本研究以斑馬魚為模式,建立一誘導性之不孕控制平台技術,首先建構表現質體pT2-ZP3-KillerRed,此質體可經由卵母細胞專一性zona pellucida 3 (zp3)啟動子表現一光敏蛋白KillerRed,其可在特定波長光線照射下產生活性氧分子造成細胞損傷。表現質體與Tol2 transposase mRNA經由顯微注射至斑馬魚後,以瞬時胚期供體跳出分析(transient embryonic excision assay, TEEA)顯示注射後之斑馬魚胚胎70%表現質體嵌合至染色體,以螢光顯微鏡下觀察一個月大之F1 Tg (ZP3:KillerRed)轉殖斑馬魚,結果顯示光敏蛋白KillerRed專一性表現於性腺位置,將一個月大F2之Tg(ZP3:KillerRed)轉殖斑馬魚以波長為510~560 nm之激發光波長照射處理三小時後,KillerRed紅色螢光有逐漸減弱之趨勢,照射處理後之成魚卵巢組織結構有明顯萎縮現象,其生殖能力相較於野生型斑馬魚下降80.32%。F3之斑馬魚胚胎具有母系表現(maternal expression)所帶下的光敏蛋白KillerRed,經過激發波長照射後第3小時發現胚胎KillerRed螢光會完全消失,且在24小時內胚胎會呈現白化死亡狀態,即使在一般日光照射下胚胎也無法存活,以2,7-Dichlorofluorescin diacetate ( DCFH-DA ) 分析ROS的產生,發現顯示激發光和日光照射後1小時,胚胎呈現綠色螢光反應,證實皆有顯著活性氧分子生成。本研究以光照方式開發基因轉殖斑馬魚誘導性不孕平台技術,未來期望能協助基因轉殖觀賞魚產業通過生物安全評估,並做為水產養殖之保種應用。
Genetically modified (GM) fish have been investigated widely in diverse fish species for improving quality. However, commercially available GM fish are restricted except transgenic ornamental fish because of global concerns the ecological impacts including gene flow and extinction of wild natural fish stock. In this regard, infertility control is a core technology for overcoming this issue. In this study, we use zebrafish to establish a platform technology of infertility control. First, we constructed an expression plasmid pT2-ZP3-KillerRed, which a KillerRed gene can be triggered by an oocyte-specific zona pellucida 3 (zp3) promoter. KillerRed is a genetically encoded photosensitizer which can generate reactive oxygen species by light to induce cell damage. In order to establish Tg(ZP3:KillerRed) zebrafish, the expression plasmid with Tol2 transposase mRNA were co-microinjected into zebrafish embryos at one-cell stage. Transient embryonic excision assay (TEEA) showed that expression plasmid in 70% of injected embryos have integrated into genome. Under the observation of fluorescence microscopy, KillerRed was expressed specifically by gonad in the F1 Tg(ZP3:KillerRed). The expression of KillerRed at 30 days post-fertilization (dpf) F2 transgenic zebrafish was declined gradually after the illumination of light for 3 hours under TRITC filter (excitation wavelength 550 nm; emission wavelength 600 nm). The light illumination mediated the gonadal atrophy in adult F2 Tg(ZP3:KillerRed), and the spawning ability of light-illuminated zebrafish was decreased 80.32% compare to that in wild type zebrafish. F3 transgenic embryo possesses maternal expression of KillerRed. The expression of KillerRed in F3 transgenic embryo was ablated after the illumination of light for 3 hours under TRITC filter, and then the embryos were dead within 24 hour even the illumination was under daylight. The generation of reactive oxygen species (ROS) in F3 transgenic embryo after 1 hour illumination were demonstrated by 2,7-Dichlorofluorescin diacetate (DCFH-DA) assay. In this study, we develop an inducible platform technology of infertility control for genetically modified zebrafish.
謝誌 I
摘要 II
Abstract IV
目錄 VI
圖目錄 IX
壹、文獻回顧 1
一、魚類基因轉殖發展 1
三、魚類傳統不孕防治技術 3
(一)、物理性防治 3
(二)、多倍體不孕 3
(三)、基因轉殖不孕技術 4
三、斑馬魚生殖系統 5
四、Tol2轉位子系統 6
五、誘導性毒性光敏蛋白(KillerRed) 7
貳、研究動機 9
參、實驗流程 10
肆、實驗材料 11
一、實驗動物 11
二、質體 11
三、實驗宿主菌株 11
四、培養基 11
五、PBS 緩衝溶液 12
六、生物藥品和化學試劑 12
七、生物反應試劑套組(Kit) 13
八、儀器設備 13
九、設計之引子列表 15
伍、 實驗方法 16
一、 基因轉殖質體構築 16
(一)、DNA膠體電泳分析 16
(二)、DNA 回收 16
(三)、聚合酶連鎖反應(Polymerase chain reaction,PCR) 17
(四)、接合反應 17
(五)、轉型作用 17
(六)、製備小量質體 18
(七)、製備大量質體 18
二、建立卵母細胞表達光敏蛋白之基因轉殖斑馬魚 19
(一)、製備Tol2 messenger RNA 19
(二)、斑馬魚產卵方法與卵盤製作 20
(三)、拉針和磨針 20
(四)、顯微注射操作 21
(五)、Transient embryo exciseon assay (TEEA) 21
三、檢測轉殖魚基因組嵌入光敏蛋白與表現 22
(一)、Genomic DNA抽取 22
(二)、魚卵RNA抽取 23
(三)、反轉錄酶反應(Reverse transcription,RT) 24
四、建立穩定表達光敏蛋白之基因轉殖斑馬魚品系 24
五、光敏蛋白活性測試 25
(一)、光照射導致性腺與卵巢組織退化之光毒殺作用 25
(二)、生育能力測定 25
(三)、光照射導致胚胎組織死亡之光毒殺作用 26
(四)、魚胚胎組織ROS測定 26
陸、結果 28
一、 構築卵母細胞表現光敏蛋白之基因轉殖質體 28
二、 建立卵母細胞表現光敏蛋白之基因轉殖斑馬魚 28
三、 光照誘導性腺與卵巢組織退化 29
四、 卵巢結構退化造成生育能力下降 30
五、 光照誘導F3 Tg(ZP3:KillerRed)受精卵胚胎死亡 30
柒、討論 32
一、 卵母細胞表達光敏蛋白之基因轉殖斑馬魚 32
二、 光毒殺誘導性腺退化造成生育率下降 33
三、 光毒殺導致魚胚胎組織死亡 33
四、 觀賞魚控制不孕技術之重要性 34
捌、未來展望 36
玖、參考文獻 37
圖 47
附件一 58
附件二 59
作者簡介 60

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