碩士 / 國立屏東科技大學 / 生物科技系所 / 101 / Genetically modified (GM) fish have been investigated widely in diverse fish species for improving quality. However, commercially available GM fish are restricted except transgenic ornamental fish because of global concerns the ecological impacts including gene flow and extinction of wild natural fish stock. In this regard, infertility control is a core technology for overcoming this issue. In this study, we use zebrafish to establish a platform technology of infertility control. First, we constructed an expression plasmid pT2-ZP3-KillerRed, which a KillerRed gene can be triggered by an oocyte-specific zona pellucida 3 (zp3) promoter. KillerRed is a genetically encoded photosensitizer which can generate reactive oxygen species by light to induce cell damage. In order to establish Tg(ZP3:KillerRed) zebrafish, the expression plasmid with Tol2 transposase mRNA were co-microinjected into zebrafish embryos at one-cell stage. Transient embryonic excision assay (TEEA) showed that expression plasmid in 70% of injected embryos have integrated into genome. Under the observation of fluorescence microscopy, KillerRed was expressed specifically by gonad in the F1 Tg(ZP3:KillerRed). The expression of KillerRed at 30 days post-fertilization (dpf) F2 transgenic zebrafish was declined gradually after the illumination of light for 3 hours under TRITC filter (excitation wavelength 550 nm; emission wavelength 600 nm). The light illumination mediated the gonadal atrophy in adult F2 Tg(ZP3:KillerRed), and the spawning ability of light-illuminated zebrafish was decreased 80.32% compare to that in wild type zebrafish. F3 transgenic embryo possesses maternal expression of KillerRed. The expression of KillerRed in F3 transgenic embryo was ablated after the illumination of light for 3 hours under TRITC filter, and then the embryos were dead within 24 hour even the illumination was under daylight. The generation of reactive oxygen species (ROS) in F3 transgenic embryo after 1 hour illumination were demonstrated by 2,7-Dichlorofluorescin diacetate (DCFH-DA) assay. In this study, we develop an inducible platform technology of infertility control for genetically modified zebrafish.
| Identifer | oai:union.ndltd.org:TW/101NPUS5111020 |
| Date | January 2013 |
| Creators | 江潁彥 |
| Contributors | 胡紹揚 老師 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 60 |
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