國立臺灣大學分子與細胞生物學研究所蔡懷楨2006-07-252018-07-062006-07-252018-07-062005http://ntur.lib.ntu.edu.tw//handle/246246/5053Myf-5 是肌肉特異性轉錄因子之一，參與調控肌原纖維及肌肉細胞的增生和分化。目前對於調控myf-5 基因的cis-acting element 並不十分清楚，故本實驗利用基因轉殖的技術，以斑馬魚 (Danio rerio)作為實驗材料，研究參與調控myf-5 基因組織及時期專一表現的區域。在構築質體中，其中包含斑馬魚myf-5 上游調控區-9977 至-1 (-9977/-1), exon 1 (E1), intron 1 (I1), exon 2 (E2)再in frame 結合GFP cDNA [(-9977/-1)/E1/I1/E2/GFP]，以顯微注射方式將其注射至單細胞時期的斑馬魚胚胎中。結果顯示螢光在體節專一表現率僅2 %；相對地，在不含有E1/I1/E2 序列之質體 (-9977/-1)/GFP 螢光表現率卻有94 %。相同的顯微注射結果也被觀察到在-8600/-1，-2937/-1 和-290/-1 結合E1/I1/E2/GFP 的實驗中。進一步地運用連續性剔除造成不同長度的intron 片段，構築質體-2937/-1/GFP 連接intron 1 片段+502/+2503，+1174/+2503，+1768/+2503，+790/+1489，+1467/+2152，+502/+1787，+502/+1199，+502/+835 和+502/+659，經顯微注射發現，轉殖胚胎體節專一螢光表現率分別為2，90，88，87，83，12， 10，8 及83 % (質體-2937/-1/GFP 螢光表現率為84 %)，表示intron 1 中的+659/+835 序列有能力抑制螢光載體節專一的表現。並且在剔除質體(-2937/-1)/GFP/(+502/+1199)中之+660/+816 序列，注射後體節專一螢光表現率即會上升至91 %。由上述證據顯示，intron 1 的+660/+816 序列抑制了myf-5 上游調控區的表現，而且發現其抑制能力是具有位置及方向的特異性。因此，myf-5 intron 1 內+660/+816 序列，可能在斑馬魚myf-5 組織特異性及發育時期特異性表現上扮演重要角色。Myf-5 is a basic helix-loop-helix transcription factor that controls muscle differentiation. During early embryogensis, myf-5 expression is transient, somiteand stage-specific. However, the negative regulation of myf-5 is poorly understood. We constructed a plasmid [(-9977/-1)/E1/I1/E2/GFP] that contains the sequence -9977 to –1, exon 1 (E1), intron 1 (I1), and exon 2 (E2) of zebrafish (Danio rerio) myf-5 and a reporter GFP gene. This plasmid was microinjected into zebrafish zygotes. Surprisingly, the somite-specific expression rate of reporter GFP in the transgenic embryos was extremely low (2 %, n=392), compared to that of (–9977/–1)/GFP (92 %, n=210). Dramatic repression of myf-5 expression was also observed in embryos microinjected with plasmids in which the sequence –8600/-1, -2937/-1 or –290/-1 was linked to E1/I1/E2/GFP. Thus, intron 1 contains a silencer that specifically represses the activity of myf-5. Functional analysis of intron 1 showed a strong, negative, cis-regulatory element was located at +502/+835. Its function was orientation- and position- dependent. The repressive capability of this silencer was completely dependent on two core motifs, IE1 (+502/+527) and IE2 (+816/+835), and a 156-bp spanning sequence that lies between them. This is the first study to identify a novel, cis-acting silencer in intron 1 that is crucial to negatively regulating zebrafish myf-5 expression.application/pdf442985 bytesapplication/pdfzh-TW國立臺灣大學分子與細胞生物學研究所myf-5intronregulatory cis-elementssomiterepressionreporthttp://ntur.lib.ntu.edu.tw/bitstream/246246/5053/1/932313B002026.pdf