鋅指蛋白ZFP352下游標的基因的鑑定

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Title:	鋅指蛋白ZFP352下游標的基因的鑑定
Authors:	范盛玢
Contributors:	生物科技研究所
Keywords:	鋅指蛋白352 Zfp352
Date:	2012
Issue Date:	2012-10-16 10:28:41 (UTC+8)
Abstract:	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.
Appears in Collections:	[Graduate Institute of Biotechnology ] thesis

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