碩士 / 國立臺灣大學 / 漁業科學研究所 / 89 / The myf-5 is the one of muscle regulatory genes involved in the proliferation of myoblasts and differentiation of myogenic cells. Because of no suitable cell-lines available for analyzing the gene regulation of zebrafish myf-5, we used transgenic zebrafish to more fully dissect the cis-regulatory elements of upstream region in vivo. Various deletion sizes of the upstream sequence of zebrafish myf-5 fused with the GFP cDNA were constructed and microinjected with 12 a-mole into the animal pole of one-celled zebrafish embryos. Results showed that the specific-GFP expression rates of embryos injected with fragments of upstream sequence from nucleotide —1 to —2.9 kb (—1/—2.9 kb), -1/-702 bp, -1/-506 bp, -1/-290 bp, -1/-154 bp and —1/-82 bp were 83.5, 62.4, 28.2, 30.0, 8.6 and 13.9 %, respectively. However, no positive signals were shown in the survival transgenic embryos injected either with fragment of —1/-62 (n=110) or with fragment of -1/-290 bp but without containing —62/-82 sequence (n=104).To define the -82/-62 motif is required for tissue specific expression, we generated transgenic fish carrying sequence upstream of myf-5 gene —82/-62 motif fusion to minimal TATA of CMV promoter-EGFP cDNA.The EGFP signals and translocation of that could be observed at somite and its expression rate (61.54%)was higher then the control group (13%).Further more, Using sequential mutagenesis PCR to generate mutated between —82/-62 motif. The results show that —69/-62 (TCCTGGCCA) motif were an important region to effect myf-5 gene expression These evidences strongly suggest that (1)The -82/-62 may be an enhancer for controlling myf-5 gene expression (2) a —62/—82 motif is the most critical sequence for controlling the specific expression of zebrafish myf-5.In addition, after mating about 500 transgenic fish the F1 transgenic fish of myf-5 gene promoter (6.3kb) fusion with EGFP cDNA were also generated.
| Identifer | oai:union.ndltd.org:TW/089NTU00451010 |
| Date | January 2001 |
| Creators | Chia-Feng Liu, 劉家鳳 |
| Contributors | Hui-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 | 70 |
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