Establishment of Tissue-Specific EGFP Transgenic Zebrafish by Tol2 Transposon Based Gal4-VP16;UAS---Killerred Gene/Enhancer Trap Approach

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Project title

Code/計畫編號

NSC96-3111-B019-001

Translated Name/計畫中文名

利用Tol2 transposon 系統建立斑馬魚的 gene/enhancer trap轉殖魚-利用Tol2跳躍子為媒介進行Gal4-VP16;UAS---KillerRed gene/enhancer trap來建立具組織特異性表現KillerRed的基因轉殖斑馬魚

Project Coordinator/計畫主持人

Chin-Hwa Hu

Funding Organization/主管機關

National Science and Technology Council

Department/Unit

Department of Bioscience and Biotechnology

Website

https://www.grb.gov.tw/search/planDetail?id=1527174

Year

2007

Start date/計畫起

01-11-2007

Expected Completion/計畫迄

01-10-2008

Bugetid/研究經費

1300千元

ResearchField/研究領域

漁業

隨著人類基因體計畫的完成，將近4-5萬個人類基因以逐一定序完成，但是我們對於這些基因的功能的了解仍不到五分之ㄧ。為進一步了解各基因體功能，目前生物學家利用各種實驗動物模式，試圖以分子遺傳的方法進行大規模的突變基因篩選，希望能夠藉此迅速找出各基因的遺傳路徑。由於斑馬魚（zebrafish, Danio rerio）的胚胎發育時間短，生長快速，易於繁殖，且受精卵透明，易於觀察，因此常被生物學家作為研究脊椎動物胚胎發育的模式物種。同時近年來隨著基因定序技術的發展，斑馬魚的基因體定序工作逐漸完成，更使斑馬魚逐漸成為最廣為使用的動物模式之ㄧ。本計畫的主要研究目的是想藉由脊椎動物的基因跳躍子Tol2 transposon 構築Gal4-VP16;UAS:KillerRed gene/enhancer trap系統，製備能在不同器官組織表現KillerRed的基因轉殖魚做為工具來進行斑馬魚胚胎發育與遺傳的相關研究。當Tol2跳躍子隨機嵌入基因體內，將藉由GAL4驅動UAS所連接之KillerRed基因表現，使其出現組織專一性的紅色螢光，續以綠光照射進行photoablation，造成KillerRed表現之細胞內產生大量ROS而凋亡，藉此專一性消除局部細胞，以觀察胚胎內不同細胞間之相互關係。此外亦將待組織分化完成後再行毒殺細胞，藉以觀察組織再生之能力。 "Although the human genome has been sequenced completely, the functions of most part of these genomes still remain unclear. Scientists have tried to declare the functions and genetic pathways of these genomes through molecular genetic approach by using several animal models. Zebrafish (Danio rerio) has been one of the most popular experimental animal model on both of genetic and developmental biology study. In addition, the whole genome of zebrafish has been sequenced over 80%. We need to develop a powerful genetic tool to reveal the functions and genetic networks of these genomes. In this project, we would like to construct a Tol2 transposon-based GAL4-VP16;UAS:KillerRed binary gene/enhancer trap system to establish a transgenic zebrafish database. The Tol2 transposition system will distribute a self-reporting gene/enhancer trap vector efficiently throughout the zebrafish genome. The vector uses the potent, hybrid transcription factor GAL4-VP16 to activate expression from a UAS:KillerRed reporter cassette. The transcriptional activity of the traped gene will be traced by the red fluorescence light. The cells with KillerRed protein will be further ablated by photobleaching. It will help us to understand the functions of ablateched cells in embryonic devekioment. In addition, these transgenic lines containing tissue-specific KillerRed expression established in this component project can cross with transgenic fish lines containing tissue-specific green an red fluorescent protein expression established respectively by either Tol2 transposon based Gal4-VP16;UAS:DsRed from the component project 2 or Gal4-VP16;UAS:EGFP vector from the component project 3 to perform double-labeling experiments."

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Establishment of Tissue-Specific EGFP Transgenic Zebrafish by Tol2 Transposon Based Gal4-VP16;UAS---Killerred Gene/Enhancer Trap Approach

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