Tissue-specific Analysis of CCN1/CYR61 Gene Promoter of Zebrafish by Transgenic Assay with Green Fluorescent Protein (GFP) Reporter Gene

Cysteine-rich 61(Cyr61/CCN1)是一種分泌性的基殖細胞蛋白(matricellular protein)，屬於CCN(Cyr61，CTGF，NOV) 家族的一員。其具有許多生物性功能，包括提供細胞的附著、誘導細胞的移動、增加生長因子誘導的細胞分裂以及促進細胞的存活，另外也有人發現胚胎的發育、血管的生合成、軟骨的生合成、腫瘤的形成以及傷口的癒合也與其存在與否有關，而且CCN1/Cyr61在很多組織都有表現，所以可以合理的推測CCN1/Cyr61對於生物體的正常發育扮演了非常重要的角色。本論文主要的目的就是在研究CCN1/Cyr61基因啟動子順式調控因子(cis-regulatory element)的功能。 為了分析此基因啟動子之活性及其調控區，我先以PCR及限制酵素方法從斑馬魚的基因體中截取出包含CCN1/Cyr61基因轉錄起點至保留序列的上游片段，將其與綠色螢光蛋白報導基因(EGFP, enhanced green fluorescent protein)鍵結形成結構體。在由大(-2902/+29)至小(-140/+29)的斑馬魚啟動子片段在過渡性的轉殖實驗中，將這些結構體以顯微注射的方式注入斑馬魚之受精卵中。結果發現大片段的結構體(-2902/+29)可驅使報導基因綠色螢光蛋白在受精後斑馬魚胚胎的脊索、體節、心臟、後腦、鰭條、上下顎軟骨組織以及表皮組織內有組織專一性的表現，另外在短片段的結構體(-140/+29)僅可在脊索、體節、心臟、後腦以及表皮組織觀察到綠色螢光，由此證明斑馬魚CCN1/Cyr61啟動子(-140/+29)之部分即含有部分可驅使CCN1/Cyr61基因在脊索、體節、心臟、後腦以及表皮組織表現的序列。 另一方面，將帶有(-2902/+29)斑馬魚CCN1/Cyr61啟動子片段之F0成魚，與野生型之成魚交配，可得F1子代，從中篩選到1個轉殖恆定品系(transgenic stable line)。其胚胎在受精72小時發育後，可明顯觀察到在前述各組織都有綠色螢光蛋白之表現，與原位雜合反應偵測內生性CCN1/Cyr61 mRNA表現的組織位置相符，這明確地證實斑馬魚CCN1/Cyr61上游(-2902/+29)之區域內即包含有大部分調控此基因表現之順式調控因子。 接著，我從基因庫中找出包含人類、小鼠、大鼠、狗、黃牛、非洲爪蛙、斑馬魚和河豚等八個物種CCN1/Cyr61基因上游的基因體DNA片段，將這些片段比對後發現在各物種的上游皆包含有數段高度保留的序列；此外，將包含斑馬魚CCN1/Cyr61啟動子高度保留片段(-2299/-2123)先利用TRANSFAC軟體分析發現，此片段中的確具有數個可能的轉錄因子結合位，進一步將其拆解成更小的4個片段，把各片段與具有HSV-thymidine kinase(TK)基礎啟動子(basal promoter)的載體鍵結成為新的結構體，再以顯微注射的方式注入斑馬魚之受精卵。在過渡性基因轉殖實驗中，我亦發現在其中3個含有不同保留片段之結構體的轉殖斑馬魚幼魚中，亦有綠色螢光蛋白在不同組織器官表現。因此，綜合上述實驗的結果，斑馬魚CCN1/Cyr61基因啟動子上游(2.9kb)之調控區的確在脊椎動物演化過程中被保留下來，並且有部分調控因子位在保留片段當中。Cysteine-rich 61(Cyr61/CCN1), one of CCN family members, is an secretory matricellular protein with multiple biological functions, such as regulating cell adhesion, inducing cell migration, enhancing growth-factor-induced cell division and prolonging cell survival. It’s function is tightly associated with embryonic development, angiogenesis, chondrogenesis, tumor forming and wound healing. Moreover, CCN1/ Cyr61 is expressed in various tissues. Based on the above mentions, CCN1/Cyr61 may play an important role in normal development of an organism. The aim of this study is to identify the cis-regulatory elements of CCN1/Cyr61 gene. I cloned and constructed its promoter DNA fragments in EGFP1 vector. Those constructs were microinjected into zebrafish zygote for their functional assay. The transient transgenic assay reveals that the longest promoter fragment (-2902/+29) could induce EGFP expression in embryo’s notochord、somite、heart、hindbrain、fin、mandible (pharyngeal arches) and epithelium; on the other hand, the proximal region (-140/+29) could only induce EGFP expression in notochord、somite、heart、hindbrain and epithelium. Besides, I obtain a transgenic stable line (F1 generation) with longest promoter fragment (-2902/+29). The expression of green fluorescent protein can be observed in the previous described tissues, which corresponds with the endogenous one of Cyr61. Therefore, I demonstrated that the upstream region (-2902/+29) of zebrafish CCN1/Cyr61 gene harbors most of its cis-regulatory elements. Comparing the upstream genomic DNA sequences of CCN1/Cyr61 from eight different animal species led to the identification of a highly conserved region (-2299~-2123) in the upstream promoter region, in which several putative transcription factor binding sites are located by TRANSFAC software analysis. Furthermore, I divided it into 4 smaller fragments and constructed each with HSV-thymidine kinase basal promoter. The transient transgenic experiments show that each different conserved sub-region has differential EGFP expression patterns. Together, the cis-regulatory elements in the upstream 2.9kb promoter region of zebrafish CCN1/Cyr61 gene is functionally conserved through vertebrates’ evolutionary process.口試委員會審定書……………………………………………………i 誌謝……………………………………………………………………ii 中文摘要………………………………………………………………iii 英文摘要………………………………………………………………v 壹、前言………………………………………………………………1 貳、實驗材料…………………………………………………………29 參、實驗方法…………………………………………………………36 肆、結果………………………………………………………………50 伍、討論………………………………………………………………57 陸、圖表………………………………………………………………61 參考文獻………………………………………………………………9