博士 / 國立臺灣大學 / 漁業科學研究所 / 91 / Mosaic expression of transgenes in the F0 generation severely hinders the study of transient expression in transgenic fish. Although many approaches have been tried to overcome these limitations, they were only marginally successful at reducing mosaicism in the F0 generation. Thus, we need a simple and effective method for enhancing uniform expression of transgenes in the F0 generation, and preventing the silencing and unstable transmission of transgenes in subsequent generations of transgenic fish. Inclusion of inverted terminal repeats (ITRs) from adeno-associated virus (AAV) in plasmid DNA significantly increased the efficiency and specificity of transgene expression in Xenopus embryos. Whether AAV-ITRs could also enhance expression and permit stable transmission of transgenes in zebrafish were evaluated in this study. Results demonstrated that the transient expression and stable transmission of transgenes are enhanced by AAV-ITRs in zebrafish. Stable transgenic lines with skeletal-muscle specific (a-actin) or ubiquitous (b-actin) expression of enhanced green fluorescence protein (EGFP) reporter gene were established. Progeny inheritance test and Southern blot analysis results strongly suggest that transgenes flanked by AAV-ITRs were integrated randomly into the genome at a single locus with a concatamerized multiplier. Thus, incorporating AAV-ITRs into transgenes results in uniform gene expression in the F0 generation and stable transmission of transgenes in zebrafish.
Juvenile zebrafish are hermaphroditic, undifferentiated gonads first develop into ovary-like tissues. In female fish, the ovary-like tissues become ovaries and produce oocytes. In male fish, ovary-like tissues are degenerated and developed into testes. To better study the molecular mechanism controlling juvenile hermaphroditism in zebrafish, we establish b-actin:EGFP transgenic lines to follow the processes of transition from ovary-like tissues to testes in vivo. Based on appearance of glowing germ cells, there are three distinct groups in b-actin:EGFP transgenics. The fish in ++ group were females (44%) which had glowing germ cells as juveniles and glowing oocytes at sexual maturation. The fish in +- and -- groups were males (56%). In juvenile, +- group fish (23%), the germ cells glowed transiently, but the fish became males at sexual maturation. In contrast, in -- group fish (33%) the germ cells proliferated slowly and the germ cells and gonads never glowed. Histological analyses showed that fish in ++, +- and — groups all developed ovary-like tissues at juvenile stage. However, ovary-like tissues are actively proliferated in ++ and +- groups but slowly proliferated in -- group. This is the reason why germ cells show glowing appearance in both ++ and +-, but not in -- groups. Interestingly, the ovary-like tissues in +- group are gradually degenerated and resulted in the fading of glowing appearance when development proced. Thus, our b-actin:EGFP transgenic lines provide useful materials for studying germ cells proliferation, juvenile hermaphroditism and sex differentiation of zebrafish in a non-invasive manner.
| Identifer | oai:union.ndltd.org:TW/091NTU00451001 |
| Date | January 2002 |
| Creators | Chung-Der Hsiao, 蕭崇德 |
| Contributors | Huai-Jen Tsai, 蔡懷楨 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 85 |
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