Analysis of genes by the Tol2-mediated enhancer trap methods in zebrafish

• Main Authors: Yi-Ru Chen, 陳怡如
• Other Authors: Chang-Jen Huang
• Format: Others
• Language: zh-TW
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/48014235305915487053

碩士 === 國立臺灣大學 === 生化科學研究所 === 99 === 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.