Gene-Regulatory and Comparative Genomics Studies on the Effect of Ethanol on the Development of Zebrafish Embryo

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Project title

Code/計畫編號

NSC96-2113-M019-002-MY2

Translated Name/計畫中文名

酒精對斑馬魚胚胎發育的影響---基因調控與比較基因體學研究

Project Coordinator/計畫主持人

Wen-Shyong Tzou

Funding Organization/主管機關

National Science and Technology Council

Co-Investigator(s)/共同執行人

胡清華

Department/Unit

Department of Bioscience and Biotechnology

Website

https://www.grb.gov.tw/search/planDetail?id=1598960

Year

2008

Start date/計畫起

01-08-2008

Expected Completion/計畫迄

01-07-2009

Co-Investigator(s)

Chin-Hwa Hu

Bugetid/研究經費

788千元

ResearchField/研究領域

生物技術(理)

" Fetal Alcohol Syndrome (FAS，胎兒酒毒性症)是孕婦懷孕期間因酗酒而引致胎兒智力遲鈍和發育畸形的病症。雖然一再地教育宣導，FAS的病症仍層出不窮，為尋求積極治療必須對FAS的機制有相當的瞭解。在我們先前的基因表現生物晶片研究發現， 1.5%酒精處理後(96hpf)的斑馬魚Danio rerio胚胎基因表現總共發現上升調控2倍的基因近200個，下降調控2倍基因近300個。除了發現表現上升的基因包含常見受壓力誘導的熱休克蛋白外，也發現一些與分節、缺氧、腦部神經分化、肌肉發育、心臟收縮相關基因，而以上基因群的功能描述與我們觀察1.5%酒精處理過的斑馬魚胚胎孵化後表現型可以相互印證。在本計畫中，我們將以三年的時間探討下列幾項問題： a. 在各胚胎發育時程，酒精對於斑馬魚胚胎基因表現影響為何? b. 在胚胎發育中，酒精對於斑馬魚胚胎基因調控網路的影響為何? 我們計畫將收集胚胎內RNA進行基因體分析，以便觀察酒精對斑馬魚胚胎發育的影響。胚胎接受不同的酒精濃度(0.5%,1.0%,1.5%)處理與不同的收集RNA時間(6,12,24,48,72,96,120hpf)。我們也將會針對酒精影響胚胎發育下遊目標基因的轉錄因子結合位置進行分析工作，探討其所影響的基因調控機制。這些系統分析將告訴我們哪些基因在網路中居樞紐位置。針對這些基因，我們也計畫以基因弱化或基因救援的方式，將gene-specific morpholino RNA oligonucleotides或表現載體利用顯微注射的方式導入受精的胚胎中，觀察對FAS表現型的影響，再以定位雜交的方式分析各器官組織細胞分化的標誌基因的表現變化。繼而配合現有酒精對各種動物模型胚胎發育影響的文獻，期能得到一兼具宏觀與細緻的理解。 " " Fetal alcohol syndrome (FAS) causes a range of disorders including abnormal facial features, growth deficiencies, and central nervous system (CNS) problems of infants due to the use of alcohol during pregnancy. Despite of the continual efforts in educating women not to drink alcohol before and during pregnancy, there are still many incidences of FAS. In order to find cure of FAS, understanding of the mechanism of FAS is necessary. Previously we employed whole genome gene-expression profiling technology to discover the change of the gene expression of zebrafish (Danio reio) embryo after the treatment of 1.5% alcohol. Nearly 200 genes are found up-regulated by at least 4 fold and 300 genes down-regulated by at least 4 folds. Among those genes include the heat shock protein, and genes involved in segmentation, hypoxia, neural development, muscle development and contractility of heart. The biological processes which these genes are involved in can fit into the scope of the affected phenotype of the larva and adult zebrafish. In this study we plan to answer the following questions. a. What will be change of gene expression at different stages of embryo development of zebrafish after the treatment of alcohol? b. What will be the impact of the gene expression from the viewpoint of gene-regulatory network? We plan to profile the gene expression pattern of zebrafish embryo. We will extract RNA from zebrafish embryo at different time (6,12,24,48,72,96,120hpf) after the treatment of alcohol of different concentration (0.5%,1.0%,1.5%). Based on the gene expression profile, we will analyze the cis-regulatory elements of the target genes influenced by alcohol to build a gene-regulatory network on which the effect of alcohol on the embryonic development can be investigated. The target genes will be confirmed by RT-PCR and by whole mount in situ hybridization. We will also use gene-specific morpholino, gene rescue of target genes and in situ hybridization of the marker genes to decipher the molecular mechanism of FAS. By combining the current understanding of FAS and our studies, we will be able to provide a global and mechanistic insight into FAS. "

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Gene-Regulatory and Comparative Genomics Studies on the Effect of Ethanol on the Development of Zebrafish Embryo

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