- 學位論文
藉由綠色螢光蛋白報導基因探討CTGF基因啟動子在斑馬魚發育過程中的功能性分析
Functional Analysis of Zebrafish CTGF gene promoter by Transgenic Assay with Green Fluorescent Protein (GFP) Reporter Gene
摘要
結締組織生長因子(CTGF)是CCN家族的成員之ㄧ，是一富含半胱氨酸、分泌性、細胞外間質相關性的蛋白質，可以在不同的細胞種類中調控不同的細胞功能，包括了細胞的增生、轉移、黏附和胞外間質的生成等功能。此外，CTGF在骨骼發育上也有特殊的重要性，特別是當梅克氏軟骨發育的階段，CTGF當作是TGF-β下游的調控分子 來刺激細胞與細胞之間的交互作用和與聚集相關的基因表現。事實上，因CTGF基因上有可對TGF-β產生感應的序列，所以CTGF許多的功能都需要TGF-β的誘導方能達成。 本論文之主要目的在分析CTGF的啟動子功能。首先，從斑馬魚之基因庫複製出CTGF基因上游約3kb的啟動子DNA片段。為了了解此基因啟動子之活性及其調控區，我先比對了斑馬魚、人類和非洲爪蟾CTGF基因上游的啟動子片段序列，將含有各物種保守性區域的片段複製出來，因此設計了大(-2893~+105)至小(-64~+22)等7個不同長度的斑馬魚CTGF啟動子與綠色螢光蛋白報導基因鍵結形成的結構體 ；或是將促進子片段與HSV-thymidine kinase (TK)及綠色螢光蛋白報導基因鍵結。之後，將這些結構體以顯微注射的方式注入斑馬魚的受精卵中。 在過渡性轉殖實驗的結果中，(-2893~+105)結構體和(-1593~ -1462)-(TK)結構體可驅使報導基因綠色螢光蛋白在受精後第五天在一些特定的位置表現，例如下顎骨、頭蓋骨、角膜、心臟、體節、脊索、底板、鮨條、表皮等，這與原位雜合反應結果的組織專一性一致。此外，(-195~+22)結構體、(-64~+22)結構體和(-195~-14)-(TK)結構體也可驅使報導基因綠色螢光蛋白在受精後第五天在脊索和體節表現，但表現範圍較少。而其它的片段則無報導基因綠色螢光蛋白表現。由此證明斑馬魚CTGF上游(-1593~-1462)和近端的部分(-195~+22)促進CTGF表現。 同時我也將帶有CTGF上游(-2893~+105) 結構體斑馬魚之F0，與野生型成魚交配，可得F1子代，目前尚未得到轉殖恆定品系(transgenic stable line)。因此，綜合上述實驗結果，在脊椎動物演化過程中 ，也許只有CTGF上游(-1593~-1462)和近端片段(-195~+22)具有保守 性的功能。
並列摘要
Connective tissue growth factor (CTGF), a member of CCN family, is a cysteine-rich, secreted, extracellular matrix-associated protein that regulates diverse cellular functions in different cell types. It modulates many cellular functions, including cell proliferation, migration, adhesion, and extracellular matrix production. Evidence suggests that there is a distinctive function of CTGF in the skeletal development. For instance, during Meckel's cartilage development, CTGF acts as a down-stream molecule of TGF-β to stimulate cell-cell interactions and the expression of condensation-associated genes. Actually, TGF-β response element is located on the CTGF gene, and CTGF can exert many functions by the induction of TGF-β. The aim of this study was to analyze the zebrafish CTGF promoter. Its cognate genomic DNA fragments were amplified by polymerase chain reaction (PCR). Upstream promoter (enhancer) fragments were constructed with EGFP (enhanced green fluorescent protein) reporter gene or with HSV-tymidine kinase (TK) basal promoter and analyzed in vivo by transient transgenic assays using zebrafish embryos. Results demonstrate that the constructs of pZF-CTGF(-2893/ +105)-EGFP1 [pCTGF- EGFP1] and pZF-CTGF(-1593/-1462)-HSV-TK-EGFP1 [pCTGF-E2-TK EGFP1]can drive the specific expression of GFP in zebrafish embryo(5dpf). The expression sites include mandible, cranium, cornea, heart, somite, notochord, floor plate, fin bud, and epidermis. It is identical to in situ hybridization result. Furthermore,the proximal promoter constructs of the pZF-CTGF(-195/+22)-EGFP1 [pCTGF-P200- EGFP1], pZF-CTGF(-64/+22)-EGFP1 [pCTGF-vTATA-EGFP1] and pZF-CTGF(-195/-44)- HSV-TK-EGFP1 [pCTGF-195xTATA-TK-EGFP1] can also drive expression of GFP in zebrafish embryo(5dpf). The expression sites include notochord and somite. However, the expression area is fewer. The zebrafish transgenic stable lines were not obtained from pZF-CTGF(-2893/+105)-EGFP1 [pCTGF-EGFP1] construct yet. The results suggest that the tissue-specific regulatory elements of the zebrafish CTGF reside within the upstream conserved region (-1593/-1462) and proximal promoter region (-195/ +22), and the regulatory mechanism of CTGF may be conserved among the vertebrate species.