Development of Infertility Technology in Transgenic Fluorescent Ornamental Fish( III )

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基本資料

Project title

Code/計畫編號

MOST105-2622-B019-001-CC1

Translated Name/計畫中文名

基因轉殖螢光觀賞魚之不孕技術開發( III )

Project Coordinator/計畫主持人

Hong-Yi Gong

Funding Organization/主管機關

National Science and Technology Council

Co-Investigator(s)/共同執行人

吳金洌
胡紹揚

Department/Unit

Department of Aquaculture

Website

https://www.grb.gov.tw/search/planDetail?id=11882983

Year

2016

Start date/計畫起

01-06-2016

Expected Completion/計畫迄

31-05-2017

Bugetid/研究經費

1500千元

ResearchField/研究領域

漁業

"基因轉殖魚類的不孕控制研究是一個重要的生物安全性考量因子，且須為可誘導性方能將基因轉殖魚類傳承下來。本計劃擬利用Piwi1 基因表現在初始生殖細胞(PGC)及雌雄生殖細胞精細胞與卵細胞發育過程，而AMH 基因表現在生殖細胞周圍的體細胞精巢之Sertoli 細胞與卵巢之濾泡細胞。擬建立魚類性腺之生殖細胞專一性Piwi1 (或Vasa)啟動子與體細胞專一性AMH 基因啟動子表現NTR-EGFP 或KillerRed 誘導性毒性蛋白之基因轉殖斑馬魚及神仙魚，藉由處理基質 Mtz 或光照分別破壞初始生殖細胞及性腺之生殖細胞與周圍的體細胞Sertoli 細胞或濾泡細胞以達到完全不孕之目標。本研究第一年由神仙魚性腺中成功選殖抗穆勒氏賀爾蒙cDNA 基因與啟動子，並構築AMH 驅動硝基還原酶與綠螢光融合蛋白表現之質體pT2-psAMH-NTR-EGFP，利用Tol2 transposon 系統成功建立F1 Tg(psAMH:NTR-EGFP)之轉殖斑馬魚。第二年計畫中成功篩選出F2 之Tg(psAMH:NTR-EGFP)轉殖斑馬魚，經解剖分析觀察到綠螢光主要專一性表現於精子與卵母周圍之細胞，証實神仙魚AMH 啟動子具有組織專一性，可成功驅動硝基還原酶與綠螢光融合蛋白表現於精巢之Sertoli cell 與卵巢之granulose cell。將一個月大F2 Tg(psAMH:NTR-EGFP) 之轉殖斑馬魚以Metronidazole (Mtz)基質浸泡處理，預期可造成性腺萎縮死亡導致不孕，此實驗目前仍持續進行中。第一年選殖出斑馬魚Piwi1/Ziwi 基因5 kb 長度之生殖細胞專一性啟動子序列，搭配 NTR/Mtz 誘殺系統，第二年成功建立 Tg(Ziwi:NTR-EGFP) 基因轉殖斑馬魚品系至F2 子代。結果顯示Ziwi 5 kb 啟動子確實能夠驅動外源 NTR-EGFP 專一性表現在生殖腺細胞中，且在受精後 12 小時即可以RT-PCR 被偵測到。但是不論基因轉殖雄魚或雌魚其EGFP 螢光訊號則要等到孵化後 25 至30 天才能以螢光顯微鏡偵測到。將一個月大基因轉殖斑馬魚經過 5 mM Mtz 基質處理四週後之轉殖斑馬魚，偵測到兩個月大基因轉殖斑馬魚其 NTR-EGFP 基因表現量有下降，螢光訊號的消失，促細胞凋亡基因 Bak-1 和 Bok-1 表現量增加，均顯示 NTR/ MTZ 誘殺系統為有效的，但仍未能完全毒殺斑馬魚生殖細胞。本年度計畫中成功篩選出F2 之 Tg(psAMH:NTR-EGFP)轉殖斑馬魚，經解剖分析觀察到綠螢光主要專一性表現於精子與卵母周圍之細胞，証實神仙魚AMH 啟動子具有組織專一性，可成功驅動硝基還原酶與綠螢光融合蛋白表現於精巢之Sertoli cell 與卵巢之granulose cell。在斑馬魚F2 Tg(psAMH:NTR-EGFP)的Mtz 浸泡實驗中，以5 mM 斑馬魚浸泡處理一月齡斑馬魚一個月的時間後，雖然可以看到卵母細胞的存在，但卵母細胞有明顯未成熟的退化現象。可以發現有精子的存在，但精巢結構有明顯的退化與發育不全。擬將每2-3 天更換5mM Mtz 溶液改進為每天更換Mtz 溶液以增進處理效果達成不孕。在芝林生物科技公司協助下同時將表現質體pT2-psAMH-NTR-EGFP 以顯微注射至神仙魚，注射後神仙魚配對所產下之F1 子代，於9 個月大時觀察到精巢與卵母細胞之周圍組織中有顯著的螢光表現，成功建立與篩選出F1 Tg(psAMH:NTR-EGFP)之轉殖神仙魚，未來將以Mtz 基質浸泡處理F2 Tg(psAMH:NTR-EGFP)之轉殖神仙魚，期望造成神仙魚性腺死亡而達成不孕之目標。將斑馬魚Piwi1/Ziwi 啟動子表現質體pT2-Ziwi-NTR-EGFP 以顯微注射至神仙魚一個細胞期受精卵中。分析11 個月大Tg(Ziwi:NTR-EGFP)基因轉殖神仙魚F0 公魚，觀察到NTR-EGFP 融合蛋白之綠色螢光專一均勻表現在精巢生殖細胞，並以PCR 及RT-PCR 偵測到其基因體中 NTR-EGFP 轉基因的存在與mRNA 在精巢表現。將Tg(Ziwi:NTR-EGFP)基因轉殖神仙魚F0 母魚與野生型公魚生下之基因轉殖神仙魚F1 受精卵830 顆，其中有94 顆發綠色螢光。由F0 基因轉殖神仙魚卵巢卵母細胞表現之NTR-EGFP 可成功篩選出Tg(Ziwi:NTR-EGFP)之轉殖神仙魚F1 子代。本計畫有助於加速基因轉殖螢光斑馬魚及神仙魚通過生物安全評估，讓技術領先與創新的基因轉殖中型螢光觀賞魚早日進入國際市場。" "The technology of infertile control for transgenic fish is considered to be prominent issue for meet the GMO biosafety and this technology must be inducible; otherwise the transgenic fish can’t be inherited. In this project, we will establish transgenic zebrafish and angelfish lines, which express an inducible toxin protein nitroreductase-EGFP fusion protein or KillerRed in the primordial germ cells, different developmental stages of germ cells including spermatogonia and oogonia by Piwi1 (or Vasa) promoter or/and in gonad surrounding somatic cells including Sertoli cells and follicle cells by AMH promoter identified from zebrafish, tilapia or angelfish. The cytotoxin will destroy germ cells and surrounding somatic cells of gonads in both male and female transgenic fish and results in infertility by the treatment of Mtz substrate solution or light irradiation. The development of technology for infertile control is considered to be the core technology for ornamental fish industry. In our results of first year, AMH cDNA and promoter were cloned from angelfish (Pterophyllum scalare) gonad. F1 Tg(psAMH:NTR-EGFP) transgenic zebrafish, which nitroreductase (NTR)-EGFP specifically expressed in gonad, was established by Tol2 transposon system. The performance of this year, F2 Tg(psAMH:NTR-EGFP) transgenic zebrafish were breed and selected. The EGFP expression specifically in periphery of testis and oocyte was demonstrated by anatomic analysis. This results demonstrated AMH promoter from angelfish can revealed tissue specificity in zebrafish, and trigger NTR-EGFP expression in sertoli cell of testis and granulose cell of ovary. We expected the gonad of the F2 Tg(psAMH:NTR-EGFP) transgenic zebrafish will be atrophy and lead to infertility by Mtz immersion treatment. This part of experiment is still on going. Piwi (P-element induced wimpy testis) is specifically expressed in germ cells and plays an important role in the control of germline development and maturation. It was originally found in Drosophila. There are two homologous genes Piwi1/Ziwi and Piwi2/Zili in zebrafish with conserved function and specificity in evolution. In this study, we developed the infertility control technology of transgenic fish by using the germline-specific Piwi1 promoter to express the inducible toxin protein NTR that can destroy the development of germ cells in gonad. The 5kb germline-specific promoter of zebrafish Piwi1/Ziwi gene was cloned and combined with the NTR/Mtz system in the 1st year and transgenic zebrafish line Tg(Ziwi:NTR-EGFP) was screened and established to F2 generation in the 2nd year. To achieve the purpose of germ cell-specific killing, F2 transgenic zebrafish with Ziwi:NTR-EGFP transgene were treated and reared in 5mM Mtz freshwater for four weeks. We found Ziwi 5 kb promoter indeed expressed the foreign NTR-EGFP in germ cells and can be detected in 12 hpf by RT-PCR. However, the EGFP fluorescent signals were not observed until 25-30 dpf in both transgenic male and female fish by fluorescent microscopy. The down-regulation of NTR-EGFP expression level, disappearance of the fluorescent signal and up-regulation of the proapoptotic Bax-1 and Bok-1 genes could be induced in 2-month zebrafish treated with 5 mM Mtz from 1-month old for four weeks. These results reveal that Ziwi1-NTR-EGFP/Mtz system in transgenic zebrafish is effective but still not completely kill all germ cells. The 1-month old F2 Tg(psAMH:NTR-EGFP) transgenic zebrafish treated with 5 mM Mtz immersion for one month exhibited immature oocytes in female ovary and obvious degenerative structure with few sperms and under development in male testis. The infertility will be improved by treatment of 5 mM Mtz solution from every 2-3 day to every one day. In this study, pT2-AMH-NTR-EGFP plasmid was also injected into angelfish (Pterophyllum scalare) for establishing transgenic angelfish by the collaboration with Jy Lin Biotechnology Inc. In 9-month old F1 offspring, the EGFP expression specifically was observed in periphery of testis and oocyte. This result elucidated that F1 Tg(psAMH:NTR-EGFP) transgenic angelfish was successfully established. In the future, we expected the goal of infertile F2 Tg(psAMH:NTR-EGFP) transgenic angelfish will be performed by Mtz immersion treatment. In addition, expression plasmid pT2-Ziwi-NTR-EGFPto express NTR-EGFP driven by zebrafish Piwi1/Ziwi1 promoter were microinjected into one-cell fertilized eggs of angelfish, respectively. Analysis of 11-month F0 transgenic male angelfish Tg(Ziwi:NTR-EGFP), green fluorescence from NTR-EGFP fusion protein expression was specifically observed in germ cells of testis. Existence of NTR-EGFP transgene and mRNA expression in testis of F0 transgenic male angelfish Tg(Ziwi:NTR-EGFP) were also detected by PCR and RT-PCR. One F0 female transgenic angelfish crossed with wild-type male fish to lay 830 fertilized eggs in which 94 F1 morula-stage embryos showed green fluorescence derived from oocytes of F0 female transgenic angelfish. F1 transgenic angelfish can be obtained from these green fluorescent embryos. We expect to perform the infertility of transgenic fluorescent zebrafish and angelfish to achieve approval of biosafety assessment and facilitate the middle-sized transgenic fluorescent ornamental fish created by innovation and leading techniques of Taiwan’s research team, to swim into the international markets in the nearby future."

Keyword(s)

基因轉殖觀賞魚
不孕控制
Piwi1
抗穆勒氏賀爾蒙
transgenic ornamental fish
infertility control
AMH

DSpace CRIS

Development of Infertility Technology in Transgenic Fluorescent Ornamental Fish( III )

基本資料

Description