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研究生:吳蕙蘭
研究生(外文):Hui-Lan Wu
論文名稱:斑馬魚cad2基因5’端上游調控序列之分析
論文名稱(外文):Analysis of zebrafish cad2 gene 5’ upstream regulatory sequence
指導教授:張清風張清風引用關係黃聲蘋
指導教授(外文):Ching-Fong ChangSheng-Ping L. Hwang
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:水產養殖學系
學門:農業科學學門
學類:漁業學類
論文種類:學術論文
論文出版年:2005
畢業學年度:93
語文別:中文
論文頁數:95
中文關鍵詞:斑馬魚消化道啟動子分析homeobox genescad2
外文關鍵詞:zebrafishgutpromoter analysishomeobox genescad2
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近年來斑馬魚成為研究脊椎動物遺傳及胚胎發育的重要模式生物,一些研究報告顯示斑馬魚消化道不同器官的形成與哺乳類有相同亦有相異之處。先前實驗室爲研究斑馬魚消化道形成之分子機制選殖了一homeodomain轉錄因子,Cad2。利用全覆式原位雜合反應來觀察Cad2 mRNA在斑馬魚胚胎不同時期之表現顯示,在受精後24小時,Cad2 mRNA開始專一的表現在斑馬魚腸道內。此結果顯示斑馬魚Cad2在腸道發育後為專一表現在腸道的基因。因此本論文欲探討Cad2基因在腸道的表現是如何被調控的。
之前實驗室以Cad2 coding region為探針由斑馬魚λ genomic DNA library得到一13 kb genomic DNA含有Cad2 5’端上游區域並選殖入pBluescript載體,並將9 kb之Xho I DNA片段連接到EGFP載體,命名為Cad2 9k pEGFP-1。但將此質體以顯微注射1-cell zygote後,無法觀察到被注射的胚胎有綠色螢光表現,可能因Cad2 5’端調節區域含有部分Cad2基因之轉譯區域,導致連結之GFP蛋白質無法形成,為將Cad2基因之轉譯區域去除,使用EXO III deletion。使用EXO III deletion後得到不包含Cad2 轉譯區域的Cad2 9k pEGFP#9-78。但Cad2 9k pEGFP#9-78經注射後雖有GFP螢光表現,但GFP的表現皆侷限在24 hpf到96 hpf胚胎的表皮與肌肉而沒有在腸道表現,推測此9kb 5’端調節區域太長,或許有些repressor domain存在而導致GFP螢光無法專一在腸道表現。再次進行EXO III deletion,以每500 bp為單位挑選21個deletion constructs分別進行顯微注射。deletion constructs Cad2 9k pEGFP # 2-29 (-10107) 到Cad2 9k pEGFP # 10-3 (-2502) 之間,所注射之質體DNA皆有相同的螢光表現模式,表現皆侷限在胚胎的表皮與肌肉。當以位於-1585 bp到-290 bp之間之deletion constructs如Cad2 9k pEGFP# 6-8 (-1585)、Cad2 9k pEGFP# 11-9 (-290) 來進行顯微注射時,觀察被注射之24 hpf到72 hpf胚胎中皆無螢光表現。推測在-2502 bp到-1585 bp之間存在有基礎的啟動子區域,如TATA box等,而這些deletion constructs因為缺乏基礎啟動子區域,所以無法驅動螢光表現。推論已分析的約9 kb Cad2 5’端上游區域仍太短,缺乏可調節GFP螢光表現在腸道的DNA序列。
先前實驗室所選殖到之Cad2 13 kb genomic DNA片段,因為沒有適當的限制酶酵素剪切位置,無法直接與EGFP載體結合;因此利用coinjection之方法來探討Cad2 5’端最上游的5.3 kb是否具有可調節GFP表現在腸道的DNA序列。將線性化Cad2 9k pEGFP #11-16質體DNA及相同莫耳數之5.3 kb Not I-PpuM I DNA片段一起顯微注射入1-cell zygote,所觀察到在不同發育時期胚胎GFP螢光表現情形與單獨注射線性化Cad2 9k pEGFP #11-16質體DNA表現類似:GFP表現皆侷限在胚胎的表皮與肌肉。綜合以上實驗結果,實驗室所選殖到13 kb genomic DNA所包含的Cad2 5’端上游區域並不具有能調節GFP專一表現在腸道的DNA序列。
以北方點墨法來分析Cad2 mRNA在不同胚胎發育時期之表現情形顯示,Cad2 RNA探針可成功的在斑馬魚72 hpf胚胎以及96 hpf胚胎的全量RNA或是mRNA中雜合到三種不同大小的mRNA,分別為5.11 kb,2.17kb以及1.69kb,以5.11 kb為主要mRNA產物,與Cad2在果蠅及人類同源性基因所合成mRNA之長度比較要大的許多。此結果亦反應出斑馬魚Cad2基因調控的複雜性及與其他生物的不同。
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.
謝辭………………………………………………………………………i
中文摘要…………………………………………………………………ii
英文摘要…………………………………………………………………iv
目錄.……………………………………………………………………vi
實驗方法目錄…………………………………………………………vii
表目錄………………………………………………………………… x
圖目錄………………………………………………………………… xi
導論………………………………………………………………………1
實驗方法…………………………………………………………………19
結果…………………………………………………………………… 41
討論………………………………………………………………………50
參考文獻……………………………………………………………… 55
圖表…………………………………………………………………… 61
附錄…………………………………………………………………… 77
附圖…………………………………………………………………… 80
Amsterdam, A., Lin, S. and Hopkins, N. (1995). The Aequorea victoria green fluorescent protein can be used as a reporter in live zebrafish embryos. Dev Biol 171, 123-129.
Beck, F., Erler, T., Russell, A. and James, R. (1995). Expression of Cdx-2 in the mouse embryo and placenta: possible role in patterning of the extra-embryonic membranes. Dev Dyn 204, 219-227.
Boonanuntanasarn, S., Yoshizaki, G. and Takeuchi, T. (2003). Specific gene silencing using small interfering RNAs in fish embryos. Biochem Biophys Res Commun 310, 1089-1095.
Carvajal, J. J., Cox, D., Summerbell, D. and Rigby, P. W. (2001). A BAC transgenic analysis of the Mrf4/Myf5 locus reveals interdigitated elements that control activation and maintenance of gene expression during muscle development. Development 128, 1857-1868.
Chawengsaksophak, K., James, R., Hammond, V. E., Kontgen, F. and Beck, F. (1997). Homeosis and intestinal tumours in Cdx2 mutant mice. Nature 386, 84-87.
Culp, P., Nusslein-Volhard, C. and Hopkins, N. (1991). High-frequency germ-line transmission of plasmid DNA sequences injected into fertilized zebrafish eggs. Proc Natl Acad Sci U S A 88, 7953-7957.
Davidson, A. J., Ernst, P., Wang, Y., Dekens, M. P., Kingsley, P. D., Palis, J., Korsmeyer, S. J., Daley, G. Q. and Zon, L. I. (2003). cdx4 mutants fail to specify blood progenitors and can be rescued by multiple hox genes. Nature 425, 300-306.
De Robertis, E. M. (1994). The Homeodomain in Cell Differentiation. In Guidebook to the Homeobox gene. Double, D., Editor. University of Geneva, Geneva, Switerland. p13-16.
Dodd, A., Chambers, S. P. and Love, D. R. (2004). Short interfering RNA-mediated gene targeting in the zebrafish. FEBS Lett 561, 89-93.
Duprey, P., Chowdhury, K., Dressler, G. R., Balling, R., Simon, D., Guenet, J. L. and Gruss, P. (1988). A mouse gene homologous to the Drosophila gene caudal is expressed in epithelial cells from the embryonic intestine. Genes Dev 2, 1647-1654.
Field, H. A., Dong, P. D., Beis, D. and Stainier, D. Y. (2003a). Formation of the digestive system in zebrafish. II. Pancreas morphogenesis. Dev Biol 261, 197-208.
Field, H. A., Ober, E. A., Roeser, T. and Stainier, D. Y. (2003b). Formation of the digestive system in zebrafish. I. Liver morphogenesis. Dev Biol 253, 279-290.
Gamer, L. W. and Wright, C. V. (1993). Murine Cdx-4 bears striking similarities to the Drosophila caudal gene in its homeodomain sequence and early expression pattern. Mech Dev 43, 71-81.
Gehring, W. J. (1987). Homeo boxes in the study of development. Science 236, 1245-1252.
Gehring, W. J. (1994). A History of the homeobox. In Guidebook to the Homeobox gene. Double, D., Editor. University of Geneva, Geneva, Switerland. p3-6.
Gehring, W. J., Affolter, M. and Burglin, T. (1994). Homeodomain proteins. Annu Rev Biochem 63, 487-526.
Gordon, J. I. and Hermiston, M. L. (1994). Differentiation and self-renewal in the mouse gastrointestinal epithelium. Curr Opin Cell Biol 6, 795-803.
Her, G. M., Chiang, C. C. and Wu, J. L. (2004a). Zebrafish intestinal fatty acid binding protein (I-FABP) gene promoter drives gut-specific expression in stable transgenic fish. Genesis 38, 26-31.
Her, G. M., Yeh, Y. H. and Wu, J. L. (2004b). Functional conserved elements mediate intestinal-type fatty acid binding protein (I-FABP) expression in the gut epithelia of zebrafish larvae. Dev Dyn 230, 734-742.
Horn, J. M. and Ashworth, A. (1995). A member of the caudal family of homeobox genes maps to the X-inactivation centre region of the mouse and human X chromosomes. Hum Mol Genet 4, 1041-1047.
Horne-Badovinac, S., Rebagliati, M. and Stainier, D. Y. (2003). A cellular framework for gut-looping morphogenesis in zebrafish. Science 302, 662-665.
Iyengar, A., Muller, F. and Maclean, N. (1996). Regulation and expression of transgenes in fish -- a review. Transgenic Res 5, 147-166.
James, R., Erler, T. and Kazenwadel, J. (1994). Structure of the murine homeobox gene cdx-2. Expression in embryonic and adult intestinal epithelium. J Biol Chem 269, 15229-15237.
James, R. and Kazenwadel, J. (1991). Homeobox gene expression in the intestinal epithelium of adult mice. J Biol Chem 266, 3246-3251.
Jin, T. and Li, H. (2001). Pou homeodomain protein OCT1 is implicated in the expression of the caudal-related homeobox gene Cdx-2. J Biol Chem 276, 14752-14758.
Joore, J. (1999). Promoter analysis in zebrafish embryos. Methods Mol Biol 127, 155-166.
Ju, B., Xu, Y., He, J., Liao, J., Yan, T., Hew, C. L., Lam, T. J. and Gong, Z. (1999). Faithful expression of green fluorescent protein (GFP) in transgenic zebrafish embryos under control of zebrafish gene promoters. Dev Genet 25, 158-167.
Kaufman, T. C., Seeger, M. A. and Olsen, G. (1990). Molecular and genetic organization of the antennapedia gene complex of Drosophila melanogaster. Adv Genet 27, 309-362.
Kimmel, C. B. (1989). Genetics and early development of zebrafish. Trends Genet 5, 283-288.
Laser, B., Meda, P., Constant, I. and Philippe, J. (1996). The caudal-related homeodomain protein Cdx-2/3 regulates glucagon gene expression in islet cells. J Biol Chem 271, 28984-28994.
Lin, S. (2000). Transgenic zebrafish. Methods Mol Biol 136, 375-383.
Lickert, H. and Kemler, R. (2002). Functional analysis of cis-regulatory elements controlling initiation and maintenance of early Cdx1 gene expression in the mouse. Dev Dyn 225, 216-220.
Lohnes, D. (2003). The Cdx1 homeodomain protein: an integrator of posterior signaling in the mouse. Bioessays 25, 971-980.
Macdonald, P. M. and Struhl, G. (1986). A molecular gradient in early Drosophila embryos and its role in specifying the body pattern. Nature 324, 537-545.
Meyer, B. I. and Gruss, P. (1993). Mouse Cdx-1 expression during gastrulation. Development 117, 191-203.
Mlodzik, M. and Gehring, W. J. (1987). Expression of the caudal gene in the germ line of Drosophila: formation of an RNA and protein gradient during early embryogenesis. Cell 48, 465-478.
Mueller-Storm, H. P., Sogo, J. M. and Schaffner, W. (1989). An enhancer stimulates transcription in trans when attached to the promoter via a protein bridge. Cell 58, 767-777.
Muller, F., Chang, B.-E., Albert, S., Fischer, N., Tora, L. and Strahle, U. (1999). Intronic enhancers control expression of zebrafish sonic hedgehog in floor plate and notochord. Development 126, 2103-2116.
Muller, F., Williams, D. W., Kobolak, J., Gauvry, L., Goldspink, G., Orban, L. and Maclean, N. (1997). Activator effect of coinjected enhancers on the muscle-specific expression of promoters in zebrafish embryos. Mol Reprod Dev 47, 404-412.
Pack, M., Solnica-Krezel, L., Malicki, J., Neuhauss, S. C., Schier, A. F., Stemple, D. L., Driever, W. and Fishman, M. C. (1996). Mutations affecting development of zebrafish digestive organs. Development 123, 321-328.
Pirrotta, V. (1990). Transvection and long-distance gene regulation. Bioessays 12, 409-414.
Shafizadeh, E., Huang, H. and Lin, S. (2002). Transgenic zebrafish expressing green fluorescent protein. Methods Mol Biol 183, 225-233.
Silberg, D. G., Swain, G. P., Suh, E. R. and Traber, P. G. (2000). Cdx1 and cdx2 expression during intestinal development. Gastroenterology 119, 961-971.
Solnica-Krezel, L., Schier, A. F. and Driever, W. (1994). Efficient recovery of ENU-induced mutations from the zebrafish germline. Genetics 136, 1401-1420
Stuart, G. W., McMurray, J. V. and Westerfield, M. (1988). Replication, integration and stable germ-line transmission of foreign sequences injected into early zebrafish embryos. Development 103, 403-412.
Stuart, G. W., Vielkind, J. R., McMurray, J. V. and Westerfield, M. (1990). Stable lines of transgenic zebrafish exhibit reproducible patterns of transgene expression. Development 109, 577-584.
Sumanas, S. and Larson, J. D. (2002). Morpholino phosphorodiamidate oligonucleotides in zebrafish: a recipe for functional genomics? Brief Funct Genomic Proteomic 1, 239-256.
Tam, P. P., Kanai-Azuma, M. and Kanai, Y. (2003). Early endoderm development in vertebrates: lineage differentiation and morphogenetic function. Curr Opin Genet Dev 13, 393-400.
Tamura, T., Konishi, Y., Makino, Y. and Mikoshiba, K. (1996). Mechanisms of transcriptional regulation and neural gene expression. Neurochem Int 29, 573-581.
Wallace, K. N. and Pack, M. (2003). Unique and conserved aspects of gut development in zebrafish. Dev Biol 255, 12-29.
Warga, R. M. and Nusslein-Volhard, C. (1999). Origin and development of the zebrafish endoderm. Development 126, 827-838.
Wells, J. M. and Melton, D. A. (1999). Vertebrate endoderm development. Annu Rev Cell Dev Biol 15, 393-410.
鄭珮宜,2003。斑馬魚Cdx2基因與消化道及軟骨性骨頭發育之關係。國立台灣海洋大學生物科技研究所碩士學位論文。
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