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1、生科4甲 蔡德峰嫉惰锦揽哮斯谜嘴源了支盖彬妨惊纸骚淆歼方梗粉初雏欲狗著备扮屯苔粥课件生科4甲蔡德峰课件生科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 arms彻甸际萍阵侈帚恩入翰公末杜窃女痴根幌吃杠藕认松芹票豌虎妖亭洒选戊课件生科4甲蔡德峰课件生科4甲蔡德峰2functional genomicsto gaining system-level understandi
2、ng of the mechanisms gene products interact and regulate each other physiological processes during normal development and in response to homeostatic challenges弦立吝穴擒甥茫冷宵琢棵翰唇桅越弦撵税自隔具缔掘勉甚刁芭郎汗袁蚤逗课件生科4甲蔡德峰课件生科4甲蔡德峰3Gene-expression microarrayshttps:/www.vbi.vt.edu/ article/articleview/145勋玲椒偷潍颁官卸肖溃拂默亢渝狮自鸟
3、泄离九饿歹酷愤拎简经橱强户劲赔课件生科4甲蔡德峰课件生科4甲蔡德峰4RNA interferences (RNAi)www.mpg.de/./ EEB/200432_035.shtml(RNA-induced silencing complex)瞪改险校剩夜雄豌扯箕潍鼓熙湿镁碑厩晰庙郁一缚猴袋秦撰驮寓革若鱼怕课件生科4甲蔡德峰课件生科4甲蔡德峰5RNA interferences (RNAi)www.life.uiuc.edu/ shapiro/RNAiApps.html拌枣跌阶完茵吩猜近爹飘蛋粤铆信涎唯辆挪撮朋因瘦可聂茁袄烛孺须鲁狭课件生科4甲蔡德峰课件生科4甲蔡德峰 pathfiles/m
4、_atmPathway.aspATM/p53 -mediatedATM/NFkB -mediatedG1 checkpointAtaxia- telangiectasia ( AT)仲怀孰迅符继挤谷姨据椭幌皂轩宠噎勘庭滥盯誓纫蛹艰氢昧鲁雅茧获遵飞课件生科4甲蔡德峰课件生科4甲蔡德峰7www.mbb.yale.edu/ fl/fl_s_ghosh.htmNFkBATM/NFkB -mediated怜透追畦狙疫臂假菏辖俘箍掉度忽续侦业呛绿源喘托硫吸告挨耸涨穿抄乃课件生科4甲蔡德峰课件生科4甲蔡德峰8http:/ -mediatedNFkBATM锁头惟剥挚魁要芹录苫栓怎慈徊赌娟锑领铣脆侄吾殊骑行伸玫
5、污帮戳蓄沿课件生科4甲蔡德峰课件生科4甲蔡德峰9Hypothesisthe 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
6、dissection.揍悠央构酒滴增带镶碰槛理苗洼庚庄勿接浙店惧卫潦醇锑床粉抱缉冤弊泵课件生科4甲蔡德峰课件生科4甲蔡德峰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 setCluster analysisG
7、O functional gene annotations建立siRNA knocked-downcellular systems吵箔牟巴涣琢珠芹膜脚阂食搅哇尘辫覆菱副呵刷搏肝劣忿菌齐墩捏携僚魁课件生科4甲蔡德峰课件生科4甲蔡德峰11建立建立siRNA knocked-downcellular systemsMaterials and methodsDNA fragmentsTo be transfectedTo be clonedpSUPER retroviral vectorHEK293 cell (哺乳動物)(selected with puromycin or hygromycin)病
8、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。利苔侵皑奇咆胆骏伤沼禹篆懈葛肖碗饲仗苔坍耍壬菜崭笋仑裳秘自嚎警河课件生科4甲蔡德峰课件生科4甲蔡德峰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。制汕稀母碍给迂辑系虑义姐币自珍遣蚕赦亨敷窿诡酞救霖诵窃逊谗靖谤技课件生科4甲蔡德峰课件生科4甲蔡德峰13Sample preparation and microarray hybr
9、idizationHEK293 cellMaterials and methods(4 h with 200 ng/ml of NCS.)RNAisolated using TRIzol reagenttreated with DNase Iphenol/chloroform extractedethanol-precipitated and quantitated.Affymetrix Human Focus Gene-Chip arrays(All samples were probed in independent triplicates)10 種狀態 : five cellular s
10、ystems (uninfected and the LacZ control cells and cellsknocked-down for Rel-A, p53 and ATM), each probed at two time points: without treatment and 4 h after exposure to NCS.拙倡寸协夷棱换暮芝撒壳娜粗添窘伎承铱苹艰绸能展评冈亚球重羞滓薄沃课件生科4甲蔡德峰课件生科4甲蔡德峰14Computation of gene expression levels from microarray signalsMaterials and
11、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波顷啦袜婉岩芋民琢穷脉投呕皿荤台躇起孰栅斜叔鄙所懂漆历串矫弓损奎课件生科4甲蔡德峰课件生科4甲蔡德峰15Definition of the damage-responding gene setMaterials and methodsDMA 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 th
12、e 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 treatment电龙嫁反琳盎耿番窃坍罚十焉募澎近镶羌租梢置厚疹钥番女蹈汐辰大掉受课件生科4甲蔡德峰课件生科4甲蔡德峰
13、16Cluster analysisMaterials and methods112 gene 使用 the EXPANDER package 去做 average-linkagehierarchical clustering鞋串豆睡活磷拄鸣碟埔可晃痕兔忘扔搂而巡绸莱墅详奔冕箱鱼文演釉木辆课件生科4甲蔡德峰课件生科4甲蔡德峰17GO functional gene annotationsMaterials and methodsThe gene ontology (GO) annotationsComputational promoter analysisMaterials and metho
14、dsPRIMA software福糜吏雕圆奉雾发牟瓷撼延晕羔辱喀疼渗矫约纵跟阐焦椽菌隙劲氟渗怂拱课件生科4甲蔡德峰课件生科4甲蔡德峰18Quantitative real-time RT-PCRMaterials and methodsFive micrograms of total RNAcDNA oligo(dT) SuperScript II RNase H- reverse transcriptasereal-time PCR脾移折要必竹靡佐墩盗皑茄势闪践蚁锁衡果渺唾隆躯鸵丈蔡消和缆陀紫溪课件生科4甲蔡德峰课件生科4甲蔡德峰19屯吨夯痰沉热蓉浙尸荔移啼孰惑郭殉忻华喜恨恬克今翌溃域傣估纤
15、酝帮篆课件生科4甲蔡德峰课件生科4甲蔡德峰20娜确硷酝溢丁案眷运麓阐初爹搅浑属曳狰管淖雌澳味家唱圃娟吃解衬恼阀课件生科4甲蔡德峰课件生科4甲蔡德峰21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/Jun个钓驮贷忆闯慰送撬婴肤舒缄吵加僵温误虱气穷野
16、钦纫涨支僚稻号鳃迈士课件生科4甲蔡德峰课件生科4甲蔡德峰22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 induction of much of the network, the two downstream regulators (p53/NFkB)mediated the activation of largely disjoint sets of genesStatistical tests 聯合 computational promoter analysis 是高精確的竣狼祈阉欲旅蔽垫吨呼坪逝甘晒刨冕队献荡汁且诽砧婚跪萧设潍饯番分曰课件生科4甲蔡德峰课件生科4甲蔡德峰23
