請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/32911
標題: | SCC-Cre轉殖小鼠之建立-
以人類CYP11A1基因啟動子驅動Cre重組酶的表現 Generation of SCC-Cre transgenic mice expressing Cre recombinase under control of human CYP11A1 promoter |
作者: | Hsu-Shui Wu 吳曙序 |
指導教授: | 胡孟君(Meng-Chun Hu) |
關鍵字: | CYP11A1基因,膽固醇側鏈裂解酶,Cre-loxP系統,腎上腺,性腺,精原細胞, CYP11A1,P450scc,Cre-loxP,ROSA26,adrenal gland,testis,ovary,spermatogonia, |
出版年 : | 2006 |
學位: | 碩士 |
摘要: | 類固醇荷爾蒙包括礦物皮質素、醣皮質素以及性荷爾蒙,對於多種正常生理功能的維持扮演了相當重要的角色。所有的類固醇都是由膽固醇所轉化而來的,在其轉化過程中,擔任第一個速率決定步驟的酶稱為膽固醇側鏈裂解酶,簡稱細胞色素P450scc。P450scc是CYP11A1基因的產物,由於類固醇荷爾蒙主要在腎上腺、性腺以及胎盤等器官中被合成,CYP11A1基因在這些地方的確有高度的表現。近年來研究還發現到腦部也可以生成類固醇,只是該處荷爾蒙生成基因,包括CYP11A1的表現量明顯比腎上腺還要來的低。在本論文研究中,我們利用一段人類CYP11A1基因上游約4.4 kb片段長的啟動子序列驅動Cre重組酶,並放置一小段來自雞的β-globin基因的HS4絕緣片段以阻擋染色體位置效應,以建立出「SCC-Cre轉殖小鼠」。我們將SCC-Cre轉殖小鼠藉由和帶有LacZ報導基因、並具有loxP序列的ROSA26小鼠交配,以驗證SCC-Cre轉殖小鼠的Cre重組酶活性。藉由X-gal染色方法,我們在所分析的大部份轉殖小鼠株號中,看到4.4 kb片段長的CYP11A1基因啟動子能夠驅動Cre表現在腎上腺皮質、睪丸的萊氏細胞以及卵巢內的濾泡細胞、黃體細胞和間質細胞處,而這些表現位置和內生性CYP11A1基因的表現分佈是相同的。除此之外,我們也看到Cre重組酶的活性在腦的部分區域也有表現。研究過程中我們更進一步發現,Cre重組酶也會表現在睪丸中非類固醇生成的精原細胞。藉由抗體的辨識,我們看到精原細胞上確實存在有明顯的LacZ蛋白質,甚至在下游精母細胞和精子也可以看到比較微弱的訊號。這個結果暗示著精原細胞可能具有荷爾蒙生成之潛能。總結本研究,我們成功地建立了SCC-Cre轉殖小鼠,能使Cre重組基因專一地表現於類固醇生成組織中,對於未來針對荷爾蒙生成細胞上相關基因功能之研究,將會是一項十分有力的工具。 Steroid hormones including mineralcorticoids, glucocorticoids, and sex hormones are crucial for a wide variety of physiologic functions. All steroids are converted from a common precursor, cholesterol. The first rate-limiting step of steroidogenesis is catalyzed by cytochrome P450scc, cholesterol side-chain cleavage enzyme, encoded by CYP11A1. Steroid hormones are mainly synthesized in adrenals, gonads and placenta and CYP11A1 is highly expressed in these steroidogenic organs. Recent studies have found that steroids can also be de novo synthesized in brain. However, the expression levels of steroidogenic genes including CYP11A1 in brain are much lower than those in adrenal glands. In this study, a 5’-flanking 4.4-kb region of human CYP11A1 gene was used to target Cre recombinase in transgenic mice, with two-copy HS4 elements of chicken β-globin gene to assure Cre expression and protect transgene against chromosome position effect. The transgenic mice were crossed to floxed ROSA26 reporter mice to examine the Cre activity. By X-gal staining assay, the results showed that the 4.4-kb fragment directed transgene expression in adrenal cortex, Leydig cells of testis and follicles, corpora lutea and stroma of ovary in most of lines, as endogenous Cyp11a1 expression patterns observed by in situ hybridization. In addition, Cre activity was also detected in brain tissues in strong expression lines. We further found that Cre was expressed in the spermatogonia of testis in all transgenic lines. The LacZ protein could be identified in spermatogonia by immunohistochemistry. Low amounts of endogenous P450scc protein in spermatogonia was visualized by IHC. It indicated that spermatogonia have the potential capacity for steroid hormones production. In conclusion, we established a SCC-Cre transgenic mouse line to provide a powerful system for studying the gene function in steroidogenic cell lineages. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/32911 |
全文授權: | 有償授權 |
顯示於系所單位: | 生理學科所 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-95-1.pdf 目前未授權公開取用 | 1.72 MB | Adobe PDF |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。