臺灣博碩士論文加值系統

同源箱基因(homeobox genes)是一群重要的具有轉錄調節能力的基因，此基因群可藉由轉譯出具有同源箱區的蛋白質(homeodomain proteins)來調控胚胎發育過程的細胞分化及決定細胞的命運。根據同源箱區具有的高度保留性氨基酸序列來設計退化性核酸引子(degenerated oligonucleotide primers)，以polymerase chain reaction (PCR)的方式篩選小鼠胚胎幹細胞cDNA基因庫，選殖後找到的其中一個新的同源箱基因-Eso-3 (Embryonic stem cell and ovary expressed gene-3)。將Eso-3同源箱區氨基酸序列與NCBI資料庫進行比對，發現與Iroquois同源箱次家族蛋白質具有最高的58%相似性，根據其同源箱區在α-螺旋Ⅰ及α-螺旋Ⅱ間多出三個氨基酸proline-tyrosine -proline之特性，因此將Eso-3歸類為TALE家族的新成員。利用資料庫比對分析及rapid amplification of cDNA ends實驗結果，發現Eso-3基因具有三種轉錄物，共可轉譯出兩種蛋白質。以reverse transcription-PCR (RT-PCR)進行Eso-3基因表現分析，得知此基因表現於成熟小鼠的各種組織中，主要表現於卵巢及腦。而分析不同發育時期的卵巢，發現Eso-3基因隨著卵巢組織的發育，表現量有遞增的趨勢。以原位雜交技術分析的結果顯示Eso-3表現在成熟卵巢組織的不同發育時期之濾泡細胞中。而在睪丸組織中，Eso-3基因隨著發育的成熟，表現量有下降的趨勢，以原位雜交技術分析結果顯示Eso-3基因表現於史托立細胞(sertoli cell)及精母細胞(spermatocyte)中。由以上結果推論Eso-3基因在生殖系統的發育中扮演重要的角色。除此之外，為了更近一步了解Eso-3在小鼠中的功能為何，目前已成功建立基因轉殖鼠表現Eso-3 small interference RNA (siEso-3)，以抑制Eso-3蛋白質在小鼠中的表現。以西方墨點法分析TG23 siEso-3基因轉殖鼠各組織之蛋白質萃取液，發現在大腦中Eso-3蛋白質的表現量有明顯的減少，此外，統計結果顯示siEso-3基因轉殖鼠可能有生殖或遺傳的障礙。對於siEso-3基因轉殖可能造成的生殖或遺傳方面的影響需要進一步研究。

The homeobox gene products act as transcription factors during animal developmental process.These genes encode homeodomain proteins that play a foundamental role in cellular differentiations and determination of cell fate. We performed a degenerated oligonucleotide polymerase chain reaction (PCR) to screen a mouse embryonic stem (ES) cell cDNA library and isolated several novel homeobox-containing genes. One of these genes, which we detected expressions in ES cells and ovary named as Eso-3, encodes a homeodomain protein that has the highest amino acid sequence identity (58%) to Iroquois subclass homeodomain gene product. That three extra amino acid residues, proline-tyrosine-proline, exist in the loop between alpha helixⅠand helix Ⅱ of the homeodomain, suggests that Eso-3 is a new member of TALE class homeobox genes. By a combination of techniques including rapid amplification of cDNA ends and EST database searching, we found that Eso-3 has 3 different transcripts and 2 different open reading frames. From the results of reverse transcription (RT)-PCR, we identified that Eso-3 expressed in all tested mouse tissues and predominantly expressed in ovary and brain. Results of RT-PCR also revealed that Eso-3 expressions in ovaries started at embryonic stage day 12.5 and continuing to the adult stage. The Eso-3 expressions in ovary were gradually increased during ovary development. Results of in situ hybridization showed that Eso-3 expressions in adult ovary were detected in the somatic cells of follicles, but not in the theca cells. In testis, results of RT-PCR revealed that Eso-3 expressions were gradually decreased from embryonic stage day 12.5 to the adult stage. Results of in situ hybridization showed that Eso-3 expressions were detected in sertoli cells and some spermatocytes of adult testis. Based on our finding, we suggest that Eso-3 play a role during development of the reproduction system. Furthermore, we generated gene knockdown transgenic mice expressing Eso-3 small interference RNA (siEso-3) to inhibit Eso-3 protein expressions in mice for investigating the in vivo functions of Eso-3 protein. Reduced expressions of Eso-3 protein levels in brain of TG23 siEso-3 transgenic mouse were observed by western analysis. The statistic results showed a significant decrease of fertility and/or inheritance in siEso-3 transgenic mice than wild-type mice. The relationship between knockdown of Eso-3 expression and mouse fertility and/or inheritance will need further analyses.

目錄

頁次
一. 摘要
中文摘要-------------------------1
英文摘要-------------------------3
二. 前言-----------------------------5
三. 材料與方法----------------------12
四. 結果----------------------------25
五. 討論----------------------------35
六. 圖表----------------------------44
七. 參考文獻------------------------68
八. 附錄----------------------------72
九. 附圖----------------------------77

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