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研究生:范盛玢
研究生(外文):Sheng-Bin Fan
論文名稱:鋅指蛋白ZFP352下游標的基因的鑑定
論文名稱(外文):Identification of ZFP352-targeted downstream genes
指導教授:黃秋容黃秋容引用關係
指導教授(外文):Chiu-Jung Huang
口試委員:林冠宏許惠貞
口試委員(外文):Kuan-Hung LinHui-Chen
口試日期:2012-07-26
學位類別:碩士
校院名稱:中國文化大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:83
中文關鍵詞:鋅指蛋白352
外文關鍵詞:ZFP352
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本研究室先前的研究中選殖出調控胚發育的鋅指蛋白基因,包括Rnf33、Rnf35 與Zfp352。這些基因僅短暫表現於小鼠著床前胚胎中,並在胚胎發育進入囊胚期前就永久靜默。Zfp352基因短暫表現於小鼠胚胎發育二到八個細胞期。Zfp352基因編碼區內無內含子,但在5’端的非編碼區中含一個4.3 kb的內含子。我們的研究重點在於闡明ZFP352調控的下游標的基因群。因此基因僅短暫表現於小鼠著床前胚胎中,故將Zfp352基因轉染到NIH/3T3這個細胞株內。Zfp352基因前含CMV promoter,可使Zfp352基因於細胞株中高度表現。Zfp352基因在NIH/3T3細胞株11號的表現量最高,因此將轉染Zfp352之NIH/3T3 11號細胞株的RNA委託陽明基因體中心進行微陣列分析。藉由微陣列分析的結果,找出表現量有差異的基因,再分別以生物資訊與即時定量PCR更進一步分析,確認被選出的基因於Zfp352轉染的細胞株中的表現量有提高之情形,以推論可能直接或間接受ZFP352活化的下游基因群。
Zinc finger protein genes have been cloned previously in our lab, including Rnf33, Rnf35 and Zfp352. These genes only express in a short time on the preimplantation embryo in mouse. The Zfp352 expresses temporarily in two- to eight-cell mouse embryo. The Zfp352 gene is intron-less in the coding region but carries a solitary 4.3-kb intron in the 5’-untranslated region. Our studies focused on identification of ZFP352-targeted downstream gene(s). Because this gene only expressed in a short time on the preimplantation embryo in mouse, the Zfp352 gene was transfected stably into NIH/3T3 cell lines. The Zfp352 gene is driven by a CMV promoter in the transfected cell lines. The expression level of the Zfp352 gene was higher in the stable cell lines No.11 which were chosen to perform microarray analysis in NYMU (National Yang-Ming University). Depending on microarray result, the genes with differential expression were chosen for further analysis by bioinformatics and Real-Time RT-PCR, to confirm the selected genes are activated in Zfp352 stable cell lines.
1.前言 4
1.1小鼠著床前胚胎發育 4
1.2鋅指蛋白的發現 8
1.3鋅指蛋白的功能 9
1.4鋅指蛋白基因與發育相關研究之起始 9
1.4.1 Gli族鋅指蛋白基因 10
1.4.2 Krox-20族鋅指蛋白基因 11
1.4.3 WT-1族鋅指蛋白基因 11
1.4.4 SP-1族鋅指蛋白基因 12
1.4.5 Krupple族鋅指蛋白基因 13
1.5鋅指蛋白352 (Zfp352) 14
1.6研究目的 14
2.材料與方法 16
2.1鋅指蛋白352 cDNA基因之構築 16
2.1.1聚合酶連鎖反應(Polymerase Chain Reaction, PCR) 16
2.1.2聚合酶連鎖反應產物之純化-Elution 17
2.1.3限制酶酵素反應 19
2.1.4載體與聚合酶連鎖反應產物之黏合-Ligation 20
2.1.5勝任細胞(Competent Cell)之製備 21
2.1.6大腸桿菌的轉形作用(Transformation) 22
2.1.7初步篩選-Cracking 23
2.1.8進階篩選-PCR 24
2.1.9大腸桿菌質體DNA之萃取 25
2.1.10定序分析與序列比對分析 27
2.2 NIH/3T3細胞的培養 28
2.2.1 NIH/3T3細胞培養基 28
2.2.2細胞培養 28
2.3細胞株的轉染(Transfection) 30
2.4細胞株RNA之萃取 31
2.5微陣列分析(Microarray Analysis) 34
2.6生物資訊分析 36
2.7反轉錄反應(Reverse Transcription;RT) 37
2.8即時定量聚合酶連鎖反應(Real-Time PCR) 39
3.結果 41
3.1 Zfp352 cDNA基因之構築及定序分析 41
3.2小鼠基因Zfp352在NIH/3T3細胞株之表現 42
3.3 NIH/3T3與11號微陣列分析結果 42
3.4生物資訊搜尋分析 43
3.5即時定量聚合酶連鎖反應分析 44
4.討論 46
4.1 Zfp352基因及ZFP352蛋白所活化之下游基因 46
4.2篩選出的功能性基因之探討 47
5.結論 51
6.參考文獻 52
7.圖表 70
8.附圖 78

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