生科4甲蔡德峰

1、生科4甲 蔡德峰1前言sequencing of the human - functional genomicsGene-expression microarrays and RNA interferences (RNAi)ATM/NFB and ATM/p53-mediated arms2functional genomicsto gaining system-level understanding of the mechanisms gene products interact and regulate each other physiological processes during n

2、ormal development and in response to homeostatic challenges3Gene-expression microarrays article/articleview/1454RNA interferences (RNAi) EEB/200432_035.shtml(RNA-induced silencing complex)5RNA interferences (RNAi) shapiro/RNAiApps.html6 pathATM/p53 -mediatedATM/NFkB -mediatedG1 checkpointAtaxia- tel

3、angiectasia ( AT)7 fl/fl_s_ghosh.htmNFkBATM/NFkB -mediated8ATM/NFkB -mediatedNFkBATM9Hypothesisthe combined experimental strategy of expression arrays and RNAi is indeed a powerful method for the dissection of complex transcriptional networks, and that computational promoter analysis can provide a s

4、trong complementary means for assessing the accuracy of this dissection.10MicroarrayanalysisDatabase searchComputational promoter analysis*- New candidate target genes*Adapted from Thomas Werner Biomolecular Engineering, 17: 87-94 (2001)TRANSFAC實驗流程圖Definition of the damage-responding gene setCluste

5、r analysisGO functional gene annotations建立siRNA knocked-downcellular systems11建立siRNA knocked-downcellular systemsMaterials and methodsDNA fragmentsTo be transfectedTo be clonedpSUPER retroviral vectorHEK293 cell (哺乳動物)(selected with puromycin or hygromycin)病毒載體用于siRNA表達，其優勢在于可以直接高效率感染細胞進行基因沉默的研究，避免

6、由于質粒轉染效率低而帶來的種種不便,而且轉染效果更加穩定。最適用于：已知一個有效的siRNA序列，需要維持較長時間的基因沉默。12以Western blotting 檢驗 RNAiRNAi并不能完全阻斷基因的表達，特別是表達異常高的基因。13Sample preparation and microarray hybridizationHEK293 cellMaterials and methods(4 h with 200 ng/ml of NCS.)RNAisolated using TRIzol reagenttreated with DNase Iphenol/chloroform ex

7、tractedethanol-precipitated and quantitated.Affymetrix Human Focus Gene-Chip arrays(All samples were probed in independent triplicates)10 種狀態 : five cellular systems (uninfected and the LacZ control cells and cellsknocked-down for Rel-A, p53 and ATM), each probed at two time points: without treatmen

8、t and 4 h after exposure to NCS.14Computation of gene expression levels from microarray signalsMaterials and methodsRMA method1. RMA 計算後, 信號明顯增強2. RMA 使用齊次多項式證明數據改進更好15Definition of the damage-responding gene setMaterials and methodsDMA method 取數值at least 1.5-fold in one control (either the uninfect

9、ed or the LacZ-infected cells), and at least 1.4-fold in the same direction in the other control.A total of 112 genes that were induced in both controls met thiscriterion and are referred to as the damage-induced gene set.Only seven genes met an analogous criterion for repression in response to NCS

10、treatment16Cluster analysisMaterials and methods112 gene 使用 the EXPANDER package 去做 average-linkagehierarchical clustering17GO functional gene annotationsMaterials and methodsThe gene ontology (GO) annotationsComputational promoter analysisMaterials and methodsPRIMA software18Quantitative real-time

11、RT-PCRMaterials and methodsFive micrograms of total RNAcDNA oligo(dT) SuperScript II RNase H- reverse transcriptasereal-time PCR192021討論RNAi and microarray technologies and a recently developed computational tool are powerfuloff-target effectscomputational promoter analysis was highly enriched for the binding signature of ATF2/ATF3/Jun22結論RNAi, microarrays and computational promoter analysis 對於 dissection of transcriptional networks 的研究是有力的Targeting the primary activator of a DNA damage response network, the upstream regulator(ATM) was indeed required for the indu