魚類基因轉殖及生長，發育，環境適應之研究（Ⅱ）─魚類之生長調控：SKI基因的調控及轉殖（Ⅱ）

Abstract

以水母綠螢光蛋白cDNA（GFP cDNA）為報導基因，具有容易檢測、高敏感 度、可在活體直接觀察等優點。我們先建構含有human cytomegalovirus immediate early enhancer/promoter 的GFP cDNA 片段，經Sca I 線性化後，各以不同DNA 濃 度，進行稻田魚未授精卵顯微注射實驗。結果顯示授精後12 小時（stage 15）開始 可以偵測到綠色螢光出現，表現率分別是5μg/ml 組為50%、7.5μg/ml 組為63%、 10μg/ml 組則為100%；但是GFP 胚胎在授精後24 小時（stage 19）的存活率，發 現10μg/ml 組為0，明顯較5μg/ml 組（40%）、7.5μg/ml 組（50%）低，顯示以 太高濃度強啟動子表現GFP 易影響稻田魚胚胎的正常發育。觀察7.5μg/ml 組表現 GFP 存活的胚胎，其綠色螢光至少可維持至孵化後10 天；與對照組比較，顯示在 稻田魚發育早期無內生性的綠色螢光干擾。 於是進一步為了解由吳郭魚選殖出c-ski 基因其在魚類胚胎早期的表現情形， 將c-ski基因上游3kb 建構至GFP cDNA 表現載體，然後以7.5ì@g/ml 的濃度做基 因轉殖後，在稻田魚胚胎發育至授精後12 小時（stage 15）即開始表現，進入原腸 胚期後，則專一在卵黃囊外膜表現，至授精後120 小時（stage 34）達到最高，並 持續至孵化。孵化後，隨著卵黃囊消失而減弱，至孵化後第5 天完全消失。顯示吳 郭魚c-ski基因的表現可能與維持卵黃囊組織正常生理活動有關。

Because green fluorescence protein (GFP) is a reportor gene used for in vivo detection, we constructed a GFP cDNA driven by human cytomegalovirus immediate early enhancer and promoter and microcinjected into medaka oocytes at various concentrations. Results showed that GFP was detectable starting at embryonic stage 15; the expression rates of 5μg/ml, %5μg/ml, and 10μg/ml groups were 50, 63 and 100%, respectively. However, the suvival rates of these GFP embryos after stage 19 were 40, 50 and 0%, respectively, suggesting too high DNA concentration of microinjected DNA caused abnormal development of embryos. The GFP was detectable until 10 days after hatching without disturbing by endogenous green light for embryos microinjected with 7.5μg/ml. Thus, we constructed a DNA fragment containing upstream 3 kb segment of tilapia c-ski gene fused with GFP cDNA and microinjected into medaka embryos at 7.5μg/ml. Resulets showed that GFP started to express at stage 15, but GFP was expressed exclusively at yolk sac at gastrula stage 34 and last until hatching out. When yolk sac was absorbed gradually the GFP disappeared gradually at the same time, and GFP was totally abolished at 5 days after hatching. Results suggest that ski protein of lilapia may involve in the development of yolk sac at early embryonic stag of fish.