專一性表達硝基還原酶基因於斑馬魚始基生殖細胞以抑制其性腺發育之研究

Abstract

過去數十年，全球基於增加水產養殖效率及生產量之目的，已將基因轉殖技術廣泛運用於各種經濟魚類，然而至今基因轉殖魚的商品化行銷除基因轉殖觀賞魚外仍受限制，主要原因是全球關注基因轉殖魚若流放到自然環境中可能會造成基因流佈或野生種的滅絕的議題。有鑑於此，基因轉殖魚的不孕控制成為解決此問題的重要技術。傳統魚類不孕控制技術如三倍體技術或用antisense RNA將生殖相關賀爾蒙剔除等方式均無法達成百分之百不孕之效果，此外若這些方法成功，則轉基因魚也無法維持保種，因此不孕平台的開發則須為誘導性。始基生殖細胞在魚類生殖上是卵巢或精巢之前驅細胞，本研究以斑馬魚為模式建立誘導性不孕控制平台技術，以始基生殖細胞專一性之nanos與kop啟動子分別表現NTR及螢光之融合蛋白，並分別建立Tg(Nanos:CFP-NTR)及Tg(Kop:NTR-GFP)基因轉殖魚，從大腸桿菌選殖而來之nitroreductase (NTR) 酵素能夠將無毒性的基質Mtz轉變為細胞毒素，導致細胞死亡，結果發現在一個細胞時期之胚胎及20天之幼魚時期開始持續不斷浸泡在5 mM Mtz會造成基因轉殖魚性腺發育有缺陷且生殖能力明顯下降，NTR/Mtz的系統會調控幼魚時期類卵母細胞的構造發生細胞凋亡而導致類卵母細胞的退化並發育成有缺陷的精巢，本結果提供作一模式平台，作為誘導性控制基因轉殖魚之不孕，並期望本模式平台未來在水產養殖上可提供經濟效益並應用於水產優質種苗的保種應用。

In the past decades, the transgenic fish of various species are being actively investigated worldwide for increasing efficiency of aquaculture production. However, the commercialization of genetically modified fish is restricted beside of transgenic ornamental fish. The global concern about ecological issue is genetically modified (GM) fish which may cause gene-flow and the threat of extinction of natural stock. In view of this issue, sterility control technology becomes important technology to solve this problem. Traditional sterility techniques such as polyploidy technology and knockdown of reproductive hormone by antisense RNA are not totally reliable. Furthermore, even through these sterile course are totally sterility, the fish stock can’t be inherited for maintenance; hence inducible sterile technology must be needed to develop for maintaining the fish stock. Nitroreductase (NTR), which is an enzyme with the ability to converse metronidazole substrate into cytotoxin and leading to cell death. In the present study, a 3 kb nanos promoter and a 5 kb askopos (kop) promoter were cloned and used to trigger NTR expression specifically in primordial germ cells (PGCs) which are germ cells give rise to gametes involved in reproduction. Through the consecutive immersion of 5 mM Mtz at early stage and juvenile gonad stage, the reproductive capacity of transgenic fish is reduced comparing to those without Mtz treatment. Our results also demonstrated that apoptosis of juvenile ovary mediated by NTR/Mtz system causes degeneration of juvenile ovary and lead to development of defective testis structure in all male fish. These results can provide as a model for inducible control the sterility of GM fish and we expected this model can offer economic benefits for maintenance of superior breeds in aquaculture purpose.