- 學位論文
條件式誘導綠螢光蛋白在斑馬魚心臟專一表現之Tet-on system
The regulation of GFP expression by Tet-on system in zebrafish heart
摘要
Tet-on system 是以Doxycycline（Dox）誘導系統啟動，使目標基因進行轉錄。此系統雖已應用於老鼠中，但是，在模式魚種斑馬魚尚未建立。本實驗利用zebrafish cardiac myosine light chain 2（cmlc2）啟動子驅動此系統，誘導後能在心臟專一表現綠螢光蛋白報導基因（GFP）。將此基因構築後，經基因轉殖分別得到兩株質體構築方式不同之轉殖魚品系T03及T21，分別觀察及比較此兩株轉殖魚報導基因的表現，在幼魚誘導的實驗部分：兩株轉殖魚之結果類似，在早期皆會受到卵膜的阻礙，以10 ug/ml Dox誘導轉殖品系24-hpf之胚胎，在7小時後看到綠螢光在心臟專一性出現；但去除卵膜則可於18-hpf進行誘導。經Dox誘導3-dpf之轉殖品系的胚胎，並利用Kodak 1D.3.5進行綠螢光定量分析，發現綠螢光亮度與誘導的時間及濃度具正相關：當誘導濃度（0.01、0.1、1及10 ug/ml）越高，誘導的時間越久（0至96小時），綠螢光的表現越強，誘導72小時後綠螢光亮度增加的趨勢漸緩，至誘導96小時，綠螢光趨近飽和，與誘導120小時之螢光亮度並無明顯差異。在成魚誘導部分：誘導兩個月大的T21品系之成魚，GFP在心臟專一表現之趨勢與3-dpf之幼魚誘導結果大致相同。在移除Dox實驗之部分：以10 ug/ml Dox誘導3-dpf之T21品系1小時，在移除Dox後，綠螢光持續增加至處理後三天，之後便下降，五天綠螢光已完全消失，受到Dox之調控，但T03觀察不到移除Dox後螢光下降之現象。總結以上比較，T21之質體構築方式會得到適用性較廣的轉殖魚品系。本實驗成它a在斑馬魚建立Tet-on system，預期將來對於基因弁鄋漪膍s、基因治療或是藥物篩選皆為十分有用的工具。
並列摘要
The tetracycline (Tet) –controlled system is well developed in many transgenic organisms. Yet, this system has not been reported in zebrafish. DNA construct, rtTA that is driven by cmlc2 promoter acts as an activator of TetO, was microinjected into the one-celled stage zebrafish embryos and there of germ-lines were generated. We examined the germ-line transmitted F2 embryos; we found that the increase of GFP expression was tightly dependent on the higher Dox dosage (ranged from 10, 1, 0.1 to 0.01 ug/ml) and on the longer induction time (ranged from 12, 24, 36, 48, 60, 72, 84 to 96 hr). When the 18-hpf embryos that de-chorion and the 24-hpf embryos were treated with 10 ug/ml Dox, GFP was specifically expressed in heart after 7 hr induction. When the 10-week fish were immersed in 10 ug/ml Dox, then GFP expression were expressed specifically in the heart after 24 hr induction. Meanwhile, the 3-dpf embryos were treated with 10 ug/ml Dox for 1 hr, the mean intensity of GFP increased gradually after withdrawal of Dox for 3 days. But after withdrawal of Dox for 5 days GFP signal disappeared completely. Therefore, Tet-ON system is capable of being turned down if Dox was removed. These evidences are the first report to demonstrate that the tet-on system can work in transgenic model fish and is highly potentially useful to study gene function in specific tissue at any stages whenever gene is induced.