利用基因轉殖的技術解析斑馬魚肌肉特異性調控因數 Myf - 5 之起動子

Abstract

Myf -5 是肌肉特異性轉錄因數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myf-5 基因上游區域的調控並不十分清楚，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，詳細研究myf-5 基因上游調控區的cis-acting element 。將不同長度之上游調控區 DNA 片段與綠螢光蛋白 cDNA （報導基因）相連，以顯微注射之方式在相同莫耳濃度的條件下，將其注射至單細胞時期的斑馬魚胚胎中，觀察到從-1nt 至-2937 nt （- 2937／- l ）、-702／-l 、-506／- l 、- 290／- l 、-154／- l 、-82／- l 之myf-5 基因上游序列在轉殖胚胎的綠螢光表現率分別為 83.5%,62.4%,28.2 % , 30.0 % , 8.6 %and 13.9 % ；若注射- 62／-1 片段，在 110 個（ n = 110 ）活下來的轉殖胚胎中並沒有觀察到任何正反應的訊號。同樣地，注射不含- 82/- 62 區域之上游 0.3kb 的promoter 片段，也沒有觀察到正反應的訊號（ n = 104 ）。但將-82/-62 接入CMV的minimal TATA box 之前進行顯微注射後，觀察到60％的螢光表現，且可以看到綠螢光表現在somite ，與CMV當promoter的EGFP表現很不相同，這些證據顯示 myf- 5 調控區中- 82／-62 是一個相當重要的cis-acting element ，具有影響斑馬魚組織特異性及發育時期特異性表現的能力。進一步地，利用 PCR 的方法設計在-82/-62 區域核甘酸的突變，發現位於 -69/-62 含有 TCCTGGCCA 序列對於myf-5 基因的調控有決定性的影響。另外，本實驗也在500 對轉殖魚中，得到了帶有myf-5 promoter 連接綠螢光蛋白的轉殖魚 Fl 子代，其綠螢光表現於腦、somite 、圍心腔外等處，並觀察到綠螢光表現自受精後18小時後會有逐漸減弱的現象。

The myf-5 is the one of muscle regulatory genes involved in the proliferation of myoblasts and differentiation of myogenic cells. Because of no suitable cell-lines available for analyzing the gene regulation of zebrafish myf-5, we used transgenic zebrafish to more fully dissect the cis-regulatory elements of upstream region in vivo. Various deletion sizes of the upstream sequence of zebrafish myf-5 fused with the GFP cDNA were constructed and microinjected with 12 a-mole into the animal pole of one-celled zebrafish embryos. Results showed that the specific-GFP expression rates of embryos injected with fragments of upstream sequence from nucleotide-1 to-2.9 kb (-1/-2.9 kb), -1/-702bp, -1/-506bp, -1/-290 bp, -1/-154bp and —1/-82bp were 83.5, 62.4, 28.2, 30.0, 8.6 and 13.9 %, respectively. However, no positive signals were shown in the survival transgenic embryos injected either with fragment of-1/-62 (n=110) or with fragment of -1/-290 bp but without containing-62/-82 sequence (n=104).To define the-82/-62 motif is required for tissue specific expression, we generated transgenic fish carrying sequence upstream of myf-5 gene-82/-62 motif fusion to minimal TATA of CMV promoter-EGFP cDNA. The EGFP signals and translocation of that could be observed at somite and its expression rate (61.54%) was higher then the control group (13%).Further more, Using sequential mutagenesis PCR to generate mutated between-82/-62 motif. The results show that-69/-62 (TCCTGGCCA) motif were an important region to effect myf-5 gene expression These evidences strongly suggest that (1)The -82/-62 may be an enhancer for controlling myf-5 gene expression (2) a-62/-82 motif is the most critical sequence for controlling the specific expression of zebrafish myf-5. In addition, after mating about 500 transgenic fish the Fl transgenic fish of myf-5 gene promoter (6.3kb) fusion with EGFP cDNA were also generated.