碩士 / 國立臺灣海洋大學 / 生物科技研究所 / 95 / Abstract
The p53 is a tumour suppressor protein.The ability of p53 to suppress tumorigenesis is mediated through its response to cellular stress, which culminates in cell growth arrest, apoptosis or senescence.The mdm2 (murine double minute 2 ) gene is an oncogene. In unstressed cells, p53 is maintained at low levels by the action of MDM2, an oncogenic E3 ubiquitin ligase (ubiquitin-protein ligating enzyme).The E3 ubiquitin ligase activity of MDM2 is essential to mediates p53 ubiquitination and proteasomal degradation.Gankyrin is an liver oncoprotein over-expressed in most hepatocellular carci-nomas (HCCs). Gankyrin can activate the ubiquitin protein ligase MDM2 which can ubiquitinate p53 leading to the proteasomal degradation of p53.We used liver fatty binding protein (L-FABP) promoter to establish liver-specific system expression in zebrafish. Then，combine GFP(Green Fluorescence protein).In this experiment, we use L-FABP(liver fatty acid binding protein) promoter to build a represent-specificity of liver system which co-operate GFP(green fluorescence protein) and MDM2/Gankyrin to observe the represent of MDM2 and Gankyrin in liver. We microinjected fusion DNA to wild type embryo, and use genomic DNA PCR and GFP expression to get the transgenic line（F0）. The transgenic line would propagate heterozygote（F1）, and we observed there are specificity of liver in green fluorescence protein by Fluorescence Microscopy in 3-5 days fish. It presents MDM2/Gankyrin was succeeded transgenic into zebrafish and would pass to the next generation. Then we used western blotting assay and RT-PCR to sieve the most strong expression in MDM2/Gankyrin transgenic fish, and crossed them to have the homozygote（F2）. Observing the decrease of P53 of MDM2/Gankyrin in F2 and the level of P53 denatured. So we built a system that has specificity of liver and transgenic line in oncogene, we could understand the specific gene expression. Then we could observe the early stage of zebrafish embryo, making it use for building and selecting anticancer drag.
| Identifer | oai:union.ndltd.org:TW/095NTOU5111007 |
| Date | January 2007 |
| Creators | Li-Che Chen, 陳立哲 |
| Contributors | Gour-Mour Her, 何國牟 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 87 |
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