碩士 / 國立臺灣海洋大學 / 水產養殖學系 / 93 / In the past decade, zebrafish becomes a model organism to study vertebrate genetic and development. Studies have shown that both conserved and diverged molecular mechanisms involved in zebrafish digestive tract development as compared with those of mammals. A homeodomain transcription factor, Cad2, was cloned previously in order to investigate molecular mechanisms involved in zebrafish gastrointestinal development. Results of whole mount in situ hybridization showed that Cad2 is expressed in the intestine after 24 hpf stages, indicating that Cad2 is a gut-specific gene. The main goal of this thesis is to investigate how Cad2 gut-specific expression is regulated.
A 13 kb genomic DNA containing Cad2 5’ upstream region was obtained and cloned into pBluescript vector by genomic DNA library screening using Cad2 coding region as probe. In addition, a 9 kb Xho I DNA fragment was cloned into EGFP vector and named Cad2 9k pEGFP-1. However, we can not detect any GFP expression in the embryos when injecting this plasmid into 1-cell zygote. This result may be due to the presence of partial Cad2 coding region in the 9 kb Xho I DNA fragment causing GFP not in frame. We used Exo III deletion to remove Cad2 partial coding region, resulting a plasmid named Cad2 9k pEGFP#9-78. Still, we detected GFP expression only in the epidermis and muscle of 24 hpf to 96 hpf embryos injected with Cad2 9k pEGFP#9-78. We speculated that some repressor domain may be present in the 9 kb Cad2 5’ upstream region and used Exo III deletion to generate various lengths of deletion constructs. Deletion constructs ranging from Cad2 9k pEGFP#2-29 (-10107) to Cad2 9k pEGFP# 10-3 (-2502) yielded GFP expression in the epidermis and muscles of injected embryos. Whereas injecting deletion constructs ranging from Cad2 9k pEGFP # 6-8 (-1585) to Cad2 9k pEGFP # 11-9 (-290) generated no GFP expression in injected embryos. We considered the presence of TATA box sequences within -2502 bp to -1585 bp Cad2 5’ upstream regions. Thus, we concluded that the 9 kb Cad2 5’upstream region is still too short to include gut-specific regulatory DNA element.
Previously obtained 13 kb genomic DNA fragment containing Cad2 5’ upstream region can not be ligated directly into EGFP vector due to lack of suitable restriction enzymes. Therefore, we used coinjection method to investigate possible involvement of 5.3 kb most 5’ Cad2 upstream region in regulating GFP to be expressed in the intestine. We compared GFP expression in embryos injected with either linearlized Cad2 9k pEGFP # 11-16 alone or coinjected with equal moles of 5.3 kb Not I-Ppu M I DNA fragment. We still detected GFP expression only in the epidermis and muscles of those injected embryos. Taken together, the 13 kb genomic DNA fragment containing Cad2 5’ upstream region did not include any gut-specific regulatory DNA fragment.
We also used northern blot to analyze Cad2 mRNA expression during different developmental stages. Three mRNA with sizes of 5.11 kb, 2.17 kb, and 1.69 kb can be hybridized with two different Cad2 RNA probes and 5.11 kb appeared to be the major Cad2 mRNA product. Comparing with sizes of mRNA from Drosophila and human Cad2 homologous genes, the size of Cad2 mRNA is rather large. This result reflected the complexity and difference of zebrafish Cad2 gene regulation as compared with those of homologous genes from other organisms.
| Identifer | oai:union.ndltd.org:TW/093NTOU5086055 |
| Date | January 2005 |
| Creators | Hui-Lan Wu, 吳蕙蘭 |
| Contributors | Ching-Fong Chang, Sheng-Ping L. Hwang, 張清風, 黃聲蘋 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 95 |
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