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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/34279

Title: 開發新穎分子遺傳技術用以鑑定班馬魚胚胎發育中壓力誘導凋亡(SIA)相關基因
Development of Novel Molecular Genetic Technique for Identifying Stress-Induced Apoptosis (Sia) Gene during Zebrafish Embryogenesis
Authors: 何國牟
Contributors: NTOU:Institute of Bioscience and Biotechnology
Keywords: 細胞凋亡;細胞自噬;螢光蛋白;基因轉殖斑馬魚;功能性基因體
Apoptosis;autophagy;green fluorescence protein;transgenic zebrafish model;functional genomic
Date: 2012-08
Issue Date: 2013-10-07T02:18:51Z
Publisher: 行政院國家科學委員會
Abstract: 摘要:近十年來,在發育生物學及遺傳學研究領域,斑馬魚已成為廣為使用之模式生物;有別於酵母菌或線蟲此等真核模式生物,斑馬魚為脊椎動物,具有獨立臟器,不論功能或構造,皆與人類極為相似。斑馬魚胚胎透明且於母體外發育,得以使用光學儀器等非侵入性之方式觀察,例如表現(綠)螢光蛋白於特殊組織、器官以利觀察,因此,斑馬魚模式系統為研究器官發育與遺傳變異之利器。 目前,本團隊已成功開發出特定選擇性基因捕獲系統(targeted selection gene trapper systems, TS-GT),別於傳統轉錄方式,此系統得以辨別基因誘發的複雜生物現象。TS-GT系統包含三大模組(研究計畫內容,圖一): (1) 乒乓啟動子捕獲模組(ping-pong promoter-trap, PP-PT),使基因組於轉錄過程中產生融合。 (2) 生理現象標定模組:功能性基因鑑定器  Annexin V (A5)結合綠螢光蛋白(secA5::eGFP),標示細胞凋亡(apotposis)現象。  lc3基因結合黃螢光蛋白(LC3::YFP),標示細胞自噬(autophagy)現象。  Cidea為細胞死亡誘發類DNA碎裂因子(cell-death inducing DNA fragmentation factor-like effector),結合紅螢光蛋白(Cidea::mCherry),標示脂肪油滴。 (3) 基因序列篩選模組 第一段(基因捕獲)模組中,於Gal4基因上游設置強效轉錄剪接接受位(splice acceptor site),令mRNA產生融合剪接而造成插入性誘變。其綠螢光蛋白表現量取決於其下游內源基因之表現強度。第二段模組則具有「功能性基因篩選分子(functional gene filters)」得以用於篩選突變基因功能;經第一段模組活化後,將引發產生第二階段之紅螢光訊號(內含於功能性基因篩選分子)。 此提案中,我們預期建構出一新穎之雙向基因體學技術「forverse (forward/reverse) genetic」,運用此技術得以於斑馬魚系統測試壓力誘發細胞凋亡「SIA (stress-induced apoptosis)」基因群之調控。TS-GT模組為I-SceI基底載體,I-SceI將能大幅提升TS-GT模組插入基因體之效率,以建立轉基因斑馬魚,而Gal4-VP16之表現量則依斑馬魚內源性基因調控機制所控制。目前我們已利用此模組建立超過百種不同的各色螢光蛋白表現形式。 基於上述各項突破性之優點,本團隊將以此TS-GT系統進行以下數種嶄新試驗: (1) 建立”活體基因庫”,以研究斑馬魚胚胎發育之生長與壓力反應。 (2) 此系統將得以高通量方式篩選發育調控及壓力反應基因群。 (3) 配合基因晶片或蛋白質體學等功能性基因體分析技術,找尋斑馬魚與壓力誘導細胞凋亡反應相關訊息傳導途徑。 (4) 結合各色螢光蛋白,將得以追蹤型態、性狀之改變,確認小分子控制壓力反應之基因調控功能。 (5) 此高感度之基因捕獲技術將可運用於魚類或其他模式生物之發育研究。
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. Therefore, it is extremely advantageous strategy for applying zebrafish model systems in the study of alimentary organs development and genetic variety. We successfully developed a new set of targeted selection gene trapper (TS-GT) systems that is designed for the identification of genes induced in complex biological niches where conventional transcriptomics is difficult to use. TS-GT systems generally comprise three modules: (1) a ping-pong promoter-trap (PP-PT) cassette generating genomic transcriptional fusions, (2) a set of reporter cassettes carrying the apoptotic marker, Annexin V (A5) protein, fused to green fluorescent protein (eGFP) (secA5::eGFP), autophagy maker , zebrafish lc3 , protein fused to Yellow fluorescent protein (YFP) (LC3::YFP) or the lipid droplet targeting domain of the cell death-inducing DNA fragmentation factor-like effector a (Cidea) protein fused to red fluorescent protein (mCherry) (mCherry::Cidea) and (3) the selection cassette for genomic sequence. The gene trap relies on a strong splice acceptor site upstream of the Gal4 gene in the first module, which is key to by passing normal splicing and making the insert mutagenic. The GFP expression depends on the transcription of downstream sequences of the endogenous gene. The second modules contain “functional gene filters” that screen the mutagenic gene functions and generates 2nd red florescent signals activated by first module. In this research proposal, we propose prospective novel "forverse" (forward/reverse) genetic techniques that are applicable to the zebrafish model and use the "forverse" techniques to examine the regulation of SIA (stress-induced apoptosis) gene in zebrafish models. We took TS-GT modules to generate GT lines. The TS-GT constructs is an I-SceI-based vector, designed to express Gal4-VP16 under the control of endogenous regulatory elements in the zebrafish genome. So far, over 100 stable lines were generated on the basis of their discrete and reproducible patterns of tdTomato expression. Given those promising advantages of those zebrafish GT lines, we plan to make use of this TS-GT fish lines as model systems for a series of novel experiment: First, we will be able to establish “in vivo gene libraries” for imagining the early stages of growth and stress response in fish embryos. Second, we will be able to identify in the developmentally regulated and stress genes in high-throughput way. Third, Functional genomic techniques such as DNA microarray and proteomic assay for identifying novel signaling pathways in zebrafish TS-GT stress mutants. Fourth, we can make use of these TS-GT stress mutants to combine color fluorescence image performances along with the change of phenotypic morphology and confirm functions of small molecules that control in stress response to the genes network. Fifth, we propose a new highly-sensitive gene trap approach and should apply studies of fish development and other aquatic models.
Relation: NSC101-2313-B019-003-MY3
URI: http://ntour.ntou.edu.tw/handle/987654321/34279
Appears in Collections:[生命科學暨生物科技學系] 研究計畫

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