Myostatin gene regulation and functional assay in zebrafish
| Autor: | Chiou-Yueh Lee, 李秋月 |
|---|---|
| Rok vydání: | 2010 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 98 Myostatin belongs to the transforming growth factor (TGF)-β superfamily, and is a secreted growth and differentiation factor and also a potent negative regulator of skeletal muscle development and growth. The myostatin (MSTN) null phenotype in mammals were characterized by extreme gains in skeletal muscles mass or “double muscle” as the cytokine negatively regulates skeletal muscle growth and development. Several aspects of MSTN biology in the fish were quit different from that in mammals. MSTN homologs had been identified in many different fish species. This diverse patter of expression in fish suggests that the biological actions of MSTN may not be restricted to skeletal muscle but may additionally influence other fish tissues as well. In this study, we had establish the zebrafish genomic library and obtained seven lambda clones by using the zebrafish MSTN I cDNA as probe. After subcloning, analysing and comparison sequence with the documented MSTN I cDNA, we found that Zg3-1 clone included 12.5Kb promoter region and exon I, Zg15-1 including 11.5 Kb promoter, exon I, intron I, exon II, intron II, exon III, intron III and 3’ untranslation region. To analyze the promoter activity of myostatin I gene, we ligated 7 different DNA fragments of the myostatin I gene into pGL3-basic vector and constructed plasmids pMSTN5.9K, pMSTN5.18K, pMSTN3.4K, pMSTN2.85K, pMSTN2.55K, pMSTN1.69K and pMSTN0.6K. As transfection to C2C12, ZFL (zebrafish liver cell) and NIH3T3 cells, the expression of luciferase activity was analysed. The results indicate that there were 12 E-boxes present in the 3.4Kb fragment of the zebrafish myostatin promoter, E11 and E12 E-box plays an important role in the regulation of the promoter activity in C2C12 and ZFL cells. We utilized microinjection of an antisense RNA-expressing vector to establish a myostatin gene knockdown hereditarily stable zebrafish strain with a double-muscle phenotype. Through real time-PCR and immunostaining analysis, the myostatin messenger (m)RNA and protein levels of homozygous transgenic zebrafish were only 33% and 26% those of the non-transgenic control, respectively. And the mRNA level of myogenic regulatory factor markers, such as MyoD, myogenin, Mrf4, and Myf5 were dramatically increased in myostatin-suppressed transgenic fish compared to the non-transgenic controls. Although there was no significant difference in body length, homozygous transgenic zebrafish showed 45% heavier body weights than those of non-transgenic controls. Histochemical analysis of 1-month-old fry showed that the area of the cross-section of homozygous transgenic fish muscle fiber was twice as large as that of non-transgenic controls. This is the first model zebrafish with a hereditarily stable myostatin-suppressed genotype and a double-muscle phenotype. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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