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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/9855

Title: 用新穎斑馬魚「虹彩突變種」來研究早期消化器官發育之分子遺傳模式
Using Novel Zebrafish "Iridescent Mutants" to Establish Molecular Genetic Models of Early Developing Digestive Organs
Authors: 何國牟
Contributors: NTOU:Institute of Bioscience and Biotechnology
國立臺灣海洋大學:生物科技研究所
Keywords: 螢光蛋白;消化器官;基因轉殖;斑馬魚模式;蛋白質體;功能性基因體;基因群網路調控
fluorescence protein;digestive organ;transgensis;zebrafish model;proteomic;functional genomic;regulation of genes networks
Date: 2010-08
Issue Date: 2011-06-28T07:17:43Z
Publisher: 行政院國家科學委員會
Abstract: 摘要:斑馬魚在發育生物學及遺傳學上已經是一個熱門的研究模式系統,牠不像其他的真核模 式系統如酵母菌和線蟲,雖是低等脊椎動物卻有著跟人類複雜結構類似的分離器官。更重要 的是,斑馬魚胚胎具有透明度可以讓非侵入式光學影像試劑如綠色螢光蛋白表現,在斑馬魚 不同組織中呈現螢光。消化器官(肝,膽囊,胰,胃及腸)發生機制是一具有錯綜複雜效果的變 化過程,其中包括由內胚層細胞(endodermal cells)特異化所成的細胞,接著肝細胞曾生而 成特化組織團 (bud),最後此組織團再轉化為臟器。一般哺乳類動物消化器官的發生從數周 至數月,斑馬魚消化器官的發生僅約2-5 天內容完成。因此,利用班馬魚為動物模式系統在研 究消化器官發生及遺傳變異上十分有利。 本實驗室先前已經建立有用的"彩虹"(多彩螢光)斑馬魚模式來研究消化器官的發育 (請參見先前研究成果說明),是利用基因轉殖的技術在消化器官(肝臟、膽囊、腸道與內外 胰臟)中表現多色螢光(綠、黃、紅與藍色螢光)。為了解脊椎動物消化器官早期發育的調控 基因網路,我們亦成功篩選了新穎斑馬魚的消化器官突變;這些突變斑馬魚的消化器官非來 自胚體初期內胚層的變化,而是來自幼魚體軸行成形後期(>4dpf)消化器官的缺失,例如全消 化器官缺失的GI#1-2 & edmea#1-2,肝臟缺陷的X-liver & Y-liver,膽囊消失的ghoul、腸 道閉鎖的Snake#1 及內外胰臟缺陷的Snake#2。然而,要進一步找出這些突變斑馬魚消化器 官發生變化的關鍵原因,我們已經產生一些經由在這些突變基因表現遺傳背景下的彩虹轉殖 基因斑馬魚。經由這些彩虹消化器官突變種的表現型顯示出類似早期脂肪肝、腸道閉鎖與膽 囊炎等症狀。因此,我們非常期待發現早期斑馬魚消化器官早期分子發病機制比較老鼠模式 和人類肝臟疾病有相同的表現型和功能。在利用微陣列晶片技術的一個前導研究中,我們也 鑑定出與X-liver 及snake 中肝、胰臟退化發展之28 可能dom 基因群。 到目前為止,只有一些動物模式(小鼠及大鼠)能進行直接成體活體實驗測試,由於斑馬 魚具備早期分子遺傳(胚胎)操作上的優勢; 因此,我們計畫利用此彩虹消化器官突變種 (Rdom)為模式系統及此28 可能dom 基因群,來發展一系列的新穎實驗:第一,將“消化器 官突變種"和“彩虹"基因轉殖斑馬魚完全結合,利用高階影像技術如共軛焦立體影像分析, 我們可以期望建立個別及相互早期消化器官發育模式。第二,利用分子遺傳分析如功能性基 因體技/DNA microarray 和proteomic assay,來辨認訊息傳遞路徑與其調控參與斑馬魚消化 器官的發育。第三,最後的挑戰是基於基因群網路調控在消化器官發育中的表現程度,結合 Rdom 模式系統快速大量之可篩選性,可能開啟一個獨特系統的功能性基因體平台給一些脊 椎動物消化疾病的研究。我們會利用這些Rdom 斑馬魚模式可以結合多色螢光影像表現的 優點,隨著消化器官突變表現型的改變,去證實小分子對應基因群網路調控的功能。
Abstract:The zebrafish has been a popular model system in developmental biology and genetics for decades. Zebrafish, unlike other eukaryotic model systems such as yeast and worms, are vertebrates with discrete organs that are very similar to their human counterparts in terms of their complex structure. Importantly, the transparency of the zebrafish embryo allows for noninvasive optical imaging of the expressed green fluorescent protein fusions in different zebrafish tissues. Vertebrate digestive organs (liver, gallbladder, pancreas, stomach and intestine) development is a cumulative effect of dynamic events, including the specification of endodermal cells which will become the specified cell, the proliferation of the cell to form the organ bud and the morphogenesis of the bud into an organ. Zebrafish alimentary organs development completes within 2-5 day. Therefore, it is extremely advantageous strategy for applying zebrafish model systems in the study of alimentary organs development and genetic variety. We had previously established useful “rainbow”zebrafish for dissecting the formation of digestive organs (table 1 in previous results). We showed that rainbow”zebrafish can serve as optical biosensors that can be used in high-throughput genetic screens to a wide various dom (digestive organs mutants ) mutants (their defect digestive organs including liver, pancreas, gut and gallbladder) arose from ENU mutagenesis and. The defect of digestive organs of these zebrafish mutants doesn't come from initial stage of endoderm, but the imperfection (table 2) that comes from the late stage of body stalk of larvae (>4 dpfs). For example, the imperfection of whole digestive organs mutants of, GI#1-2 & edmeas#1-2; the liver drawback mutants, X-liver& Y-liver; the gall bladder disappears in ghoul and intestinal lock in snake#1; and the pancreas defects of mutants in snake#2. However, to find out the decisive reason of these mutation occurrence, we had already produced compound rainbow transgenic zebrafish under these mutated genes background. Thus, we expect to find their molecular pathogenesis in early stage of zebrafish GI diseases displaying phenotypically or functionally equivalent to mouse models or human GI diseases. In a pilot study using the microarray techniques, we also identify some 28 candidate genes that developmentally regulate a recessive lethal mutation in X-liver and snake that alters processing of liver and pancreatic organogenesis. Given those promising advantages of those zebrafish lines and the 28 candidate dom gene profiles, we plan to make use of this Rdom (rainbow + digestive organ mutants) as model systems for a series of novel experiment: First, in using fully combined transgenic lines between “digestive organ mutants” and “rainbow” transgenic lines, we will be able to establish “in vivo” images via high level imaging technique such as confocal imaging analysis and find out the early stages of digestive organs formation. Second, many functional genomic techniques such as DNA microarray and proteomic assay for identifying signaling pathways and its regulators involved in zebrfish digestive organs development. Third, We will make use of these Rdom to combine many advantages of color fluorescence image performances along with the phenotypic change of digestive organ mutants and confirm functions of small molecules that control in response to the genes network.
Relation: NSC99-2313-B019-009-MY2
URI: http://ntour.ntou.edu.tw/ir/handle/987654321/9855
Appears in Collections:[生命科學暨生物科技學系] 研究計畫

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