利用Tol2跳躍子所衍生增強子捕捉方法分析斑馬魚基因之表現

Abstract

近年來斑馬魚已成為研究脊椎動物發育的一個重要模式生物。在過去果蠅發育基因功能的研究中，Gene trap和Enhancer trap方法是一個相當強大且被廣泛應用的工具。然而過去在脊椎動物系統上因缺乏有效率的基因轉殖技術，有很長一段時間這些方法無法被應用在斑馬魚相關研究上。一直到Tol2跳躍子的發現，結合Tol2隨機嵌入特性的Gene trap和Enhancer trap方法才被應用至斑馬魚上。藉由Tol2將這些載體帶入基因組的過程，可建立出許多具組織特異性GFP或Gal4表現的基因轉殖魚。另一方面，在果蠅上被廣泛採用的酵母菌Gal4-UAS轉錄活化系統也開始應用到斑馬魚上。受限於利用full-length Gal4構築的質體之轉殖基因表現量較低，這個系統經過後續研究的改良，發現以Gal4-VP16融合蛋白取代full-length Gal4可改善此情形。2007年Davison等人發表了一個結合Tol2,Gal4-UAS系統,gene trap和enhancer trap 的SAGVG基因轉殖載體。過去幾年本實驗室已成功地利用此SAGVG載體建立一系列在心臟、腦、眼睛、肌肉、血管、神經膠質、黑色素細胞、表皮細胞及神經軸突等具組織特異性Dendra2或DsRed表現的基因轉殖魚，本研究則從中挑選三組分別表現在中樞神經系統和血管的基因轉殖魚進行進一步的分析。 本研究的主要目的為分析SAGVG載體建立之基因轉殖魚，以鑑定其特殊螢光表現是受到哪些內生性基因的增強子或啟動子影響，並期望可以選殖出專一調控特定部位表現的啟動子。首先，利用Ligation-mediated PCR(LM-PCR)分別得到Tol2嵌入位周圍的斑馬魚基因組DNA片段，而後將這些片段定序並進行Ensembl斑馬魚基因組資料庫的比對，最後在斑馬魚染色體上定位出四個位於基因轉錄區以外的Tol2嵌入位。根據Davison提出的機制，初步推測SAGVG載體的特殊螢光表現是受到Tol2嵌入位附近具有相似表現位置的內生性基因增強子影響。接著利用zfin資料庫分析Tol2嵌入位上下游基因的表現位置，結果得到四個和基因轉殖魚有類似表現位置的預測基因。而後藉由全覆式原位雜合反應和顯微注射啟動子分析這四個預測基因的表現位置，以確認基因轉殖魚的螢光表現和預測基因表現是受到相同增強子的調控。此外，本研究也成功地利用此方法找到專一調控脊索(notochord)表現的組織特異性啟動子(tissue-specific promoter)。未來可利用此專一啟動子作為標定脊索的marker，以利日後斑馬魚胚胎發育的研究。

Zebrafish has become an important model organism for studying vertebrate development. In the past, enhancer trap and gene trap methods are powerful tools in studying the gene functions of Drosophila. However, for a long period of time, these methods had not been applied to vertebrate systems mainly due to lack of an efficient transgenesis technique. It is not until the discovery of the Tol2 transposable element that the Tol2-mediated gene trap and enhancer trap methods were applied to zebrafish. By creating random insertions of these constructs in the genome, transgenic zebrafish expressing GFP or Gal4 proteins in specific cells, tissues or organs are generated. A bipartite yeast Gal4-UAS system being used in Drosophila to establish tissue-specific gene expression lines was tested in zebrafish as well. To achieve stronger inductions of a UAS transgene, a modified Gal4-VP16 fusion gene with stronger transcriptional activities was published to replace the full-length Gal4. In 2007, Davison et al demonstrated a Tol2 transposon-based Gal4-VP16;14xUAS:eGFP gene/enhancer trap construct called SAGVG. Based on this self-reporting construct, we successfully generated several transgenic zebrafish lines showing tissue-specific Dendra2 or DsRed fluorescent expressions in the heart, brain, eyes, muscle, blood vessel, glia, melanocyte, skin, and neuronal axon. In this study, 3 transgenic lines with unique expressions in the CNS and blood vessel were selected for further analysis. The specific aim of this study is to identify the Tol2 insertions linked to Dendra2 or DsRed expression of the transgenic zebrafish created by SAGVG construct. Through this approach, we have a chance to discover novel tissue-specific promoters. By employing ligation-mediated polymerase chain reaction (LM-PCR) and DNA sequencing, we mapped 4 Tol2 insertions on the zebrafish chromosome. According to the potential mechanisms proposed by Dr. Davison, we suggested that the unique expression patterns of SAGVG construct is regulated by the enhancers of the endogenous genes because all of the Tol2 insertions are outside the transcribed region. On the other hand, by analyzing expression patterns of genes close to the Tol2 insertions with the zfin database, we obtained 4 predicted genes showing similar expression patterns to those of the transgenic zebrafish lines. Finally, analysis of the predicted genes by whole-mount in situ hybridization and promoter microinjection was carried out to further confirm that the Dendra2 or DsRed expressions are under control of the corresponding enhancer regulatory element. Moreover, we also obtained a notochord-specific promoter by using the Tol2-mediated gene/enhancer trap method. This promoter will be utilized as a notochord marker for future researches in zebrafish embryogenesis.