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標題: | 魚類基因啟動子在魚類細胞株及斑馬魚胚胎中之活性分析 Expression of Heterologous Promoter Sequence in Fish Cell Line and Zebrafish Embryos |
作者: | Chia-Ying Chu 朱家瑩 |
出版年 : | 1997 |
學位: | 碩士 |
摘要: | 為了研究細胞株及胚胎中的啟動子活性和調控機制,我們構築數種包含不同基因啟動子區域的檢測基因表現載體。使用的檢測基因為β-半乳糖?以及綠螢光蛋白(GFP)。啟動子區域則分別來自鯉魚(Cyprinus carpio)的JAK1、cdc2基因,和圓斑河魨(Tetraodon fluviatilis)的JAK1、fos、RET基因。在細胞株中的活性分析顯示,河魨JAK1基因啟動子的活性僅為正對照組CMV啟動子活性的7%;河魨fos基因啟動子較強,為CMV啟動子的15%。若以顯微注射法將各表現質體送入斑馬魚(Danio rerio)受精卵中進行活性分析,我們發現河魨JAK1啟動子有較高的表現活性,約達CMV啟動子的80%,而河魨fos基因啟動子僅為30%。在胚胎中由於具有不同的細胞種類,能提供不同的基因調控條件,因此與細胞株中的in vitro結果有所不同。分析各基因啟動子在胚胎中的作用部位,發現河魨JAK1、河魨fos、和鯉魚JAK1基因啟動子表現部位多集中於軀幹中段;河魨RET較集中於頭部;而鯉魚cdc2基因啟動子則有全身性的表現。統計不同啟動子表現的細胞種類,鯉魚cdc2基因啟動子及河魨RET基因啟動子多為表皮和頭部區域,河魨JAK1、河魨fos、鯉魚JAK1等基因啟動子則多出現於肌肉和脊索中,顯示部份的基因啟動子可能具有組織專一性的表現能力。此外,以不同長度的鯉魚cdc2基因啟動子進行活性分析,我們發現可能存在一個轉錄增強序列,該序列(5'-AAATTAACAAA-3')與已知老鼠cdc2基因的轉錄增強序列(5'-AAGTTACAAA-3')有很高的相似性。由於檢視β-半乳糖?活性時,必須犧牲胚胎進行固定染色,因此另外選用GFP作為檢測基因,追蹤觀察啟動子在活體中的表現活性。我們所構築的鯉魚JAK1啟動子GFP表現質體,能成功地表現於斑馬魚胚胎及幼魚體中,而在肌肉、脊索等部位放出綠色螢光。利用本論文在斑馬魚胚胎中偵測啟動子活性所建立的in vivo分析方式,將可應用於基因轉殖魚類的研究上,達到攜帶基因於特殊部位表現,或是全身性強而有效表現等目的。 In order to study the gene regulation in cell lines and embryos, several expression vectors containing different heterologous promoters as well as lacZ reporter gene were constructed. The promoter regions were obtained from a variety of fish genes including common carp (Cyprinus carpio) JAK1 and cdc2 genes as well as the round-spotted pufferfish (Tetraodon fluviatilis) JAK1, RET and c-fos genes. The results of promoter assay were different in cell lines and in embryos. When transfected into carp CF cell line, pf-fos promoter displayed strong lacZ activity, about 15% of that of pCMV β, While pf-JAK1 promoter had only 7% of that of pCMV β. When these constructs were injected into zebrafish (Danio rerio) embryos at one-cell-stage, the pf-JAK1 promoter displayed stronger expression activity than that of pf-fos promoter. Analyzing the expression pattern of 24 -h (hours after fertilization at 28℃) embryos, the β-gal-expressed cells of pf-JAK1 and c-JAK1 promoters were predominantly located in notochord and muscle in the trunk segments of embryos, whereas those of c-cdc2 promoter were located ubiquitously. The majority of expressing cells of pf-RET promoter was located in the head and skin region. We also performed deletion analysis of c-cdc2 promoter and a positive regulation region between -211 and -146 was defined. A sequence in the region (5'-AAATTAACAAA-3') has high similarity with rat cdc2 enhancer element (-276AAGTTACAAA-267). With the goal of detecting gene expression in the embryo at different developmental stages, the green fluorescent protein (GFP) from the jellyfish (Aequorea victoria) was chosen as reporter gene. GFP fluorescence can be observed non-invasively in living cells without any substrate or cofactor. A GFP expression vector driven by c-JAK1 promoter was constructed and microinjected into zebrafish embryos. At 44-h and 72-h after injected, the green fluorescence was visible in muscle and notochord. Our data indicated that the transient expression system, by means of transgenic zebrafish, could be used as rapid analysis of promoter activity and gene regulation in vivo. Utilizing this system, we found that some promoters were expressed ubiquitously whereas other promoters were expressed at restricted area. Thus, analysis of promoter activity would be useful for further studies. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/76297 |
全文授權: | 未授權 |
顯示於系所單位: | 動物學研究所 |
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