课件生科4甲蔡德峰.ppt

1,生科4甲 蔡德峰,2,前言,sequencing of the human - functional genomics Gene-expression microarrays and RNA interferences (RNAi) ATM/NFB and ATM/p53-mediated arms,3,functional genomics,to gaining system-level understanding of the mechanisms gene products interact and regulate each other physiological processes during normal development and in response to homeostatic challenges,4,Gene-expression microarrays,/ article/articleview/145,5,RNA interferences (RNAi),www.mpg.de/./ EEB/200432_035.shtml,(RNA-induced silencing complex),6,RNA interferences (RNAi),/ shapiro/RNAiApps.html,7,/ pathfiles/m_atmPathway.asp,ATM/p53 -mediated ATM/NFkB -mediated,G1 checkpoint,Ataxia- telangiectasia ( AT),8,/ fl/fl_s_ghosh.htm,NFkB,ATM/NFkB -mediated,9,/popup/cbc/NFKB_Interactive_Pathway.htm,ATM/NFkB -mediated,NFkB,ATM,10,Hypothesis,the 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 strong complementary means for assessing the accuracy of this dissection.,11,Microarray analysis,Database search,Computational 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 set,Cluster analysis,GO functional gene annotations,建立siRNA knocked-down cellular systems,12,建立siRNA knocked-down cellular systems,Materials and methods,DNA fragments,To be transfected,To be cloned,pSUPER retroviral vector,HEK293 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。,13,以Western blotting 檢驗 RNAi,RNAi并不能完全阻斷基因的表達，特別是表達異常高的基因。,14,Sample preparation and microarray hybridization,HEK293 cell,Materials and methods,(4 h with 200 ng/ml of NCS.),RNA,isolated using TRIzol reagent treated with DNase I phenol/chloroform extracted ethanol-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 cells knocked-down for Rel-A, p53 and ATM), each probed at two time points: without treatment and 4 h after exposure to NCS.,15,Computation of gene expression levels from microarray signals,Materials and methods,RMA method,1. 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,16,Definition of the damage-responding gene set,Materials and methods,DMA method 取數值at least 1.5-fold in one control (either the uninfected 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 this criterion and are referred to as the damage-induced gene set. Only seven genes met an analogous criterion for repression in response to NCS treatment,17,Cluster analysis,Materials and methods,112 gene 使用 the EXPANDER package 去做 average-linkage hierarchical clustering,18,GO functional gene annotations,Materials and methods,The gene ontology (GO) annotations,Computational promoter analysis,Materials and methods,PRIMA software,19,Quantitative real-time RT-PCR,Materials and methods,Five micrograms of total RNA,cDNA,oligo(dT) SuperScript II RNase H- reverse transcriptase,real-time PCR,20,21,22,討論,RNAi and microarray technologies and a recently developed computational tool are powerful off-target effects computational promoter analysis was highly enriched for the binding signature of ATF2/ATF3/Jun,23,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 requi