生科4甲蔡德峰ppt課件

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

2、 normal development and in response to homeostatic challengesGene-expression / article/articleview/145RNA interferences (RNAi)mpg.de/./ EEB/201932_035.shtml(RNA-induced silencing complex)RNA interferences (RNAi)/ shapiro/RNAiApps.htmlbiocarta/ pathfiles/m_atmPa

3、thway.aspATM/p53 -mediatedATM/NFkB -mediatedG1 checkpointAtaxia- telangiectasia ( AT)/ fl/fl_s_ghosh.htmNFkBATM/NFkB -mediatedemdbiosciences/popup/cbc/NFKB_Interactive_Pathway.htmATM/NFkB -mediatedNFkBATMHypothesis the combined experimental strategy of expression arrays and RNAi is indee

4、d 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.MicroarrayanalysisDatabase searchComputational promoter analysis*- New candidate target genes*Ad

5、apted from Thomas Werner Biomolecular Engineering, 17: 87-94 (2019)TRANSFACTRANSFAC實(shí)驗(yàn)流程圖Definition of the damage-responding gene setCluster analysisGO functional gene annotations建立siRNA knocked-downcellular systems建立建立siRNA knocked-downcellular systems Materials and methodsDNA fragmentsTo be transfe

6、ctedTo be clonedpSUPER retroviral vectorHEK293 cell (哺乳動(dòng)物)(selected with puromycin or hygromycin)病毒載體用于siRNA表達(dá)，其優(yōu)勢(shì)在于可以直接高效率感染細(xì)胞進(jìn)行基因沉默的研討，防止由于質(zhì)粒轉(zhuǎn)染效率低而帶來(lái)的種種不便,而且轉(zhuǎn)染效果更加穩(wěn)定。最適用于：知一個(gè)有效的siRNA序列，需求維持較長(zhǎng)時(shí)間的基因沉默。以Western blotting 檢驗(yàn) RNAiRNAi并不能完全阻斷基因的表達(dá)，特別是表達(dá)異常高的基因。Sample preparation and microarray hybridizati

7、onHEK293 cell Materials 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(tài) : five cellular systems

8、 (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.Computation of gene expression levels from microarray signals Materials and methodsRMA method1. RMA 計(jì)算後, 信號(hào)明顯增強(qiáng)2. RMA 運(yùn)用齊次多項(xiàng)式證明數(shù)據(jù)改進(jìn)更好

9、Definition of the damage-responding gene set Materials and methodsDMA method 取數(shù)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 thiscriter

10、ion and are referred to as the damage-induced gene set.Only seven genes met an analogous criterion for repression in response to NCS treatmentCluster analysis Materials and methods112 gene 運(yùn)用 the EXPANDER package 去做 average-linkagehierarchical clusteringGO functional gene annotations Materials and m

11、ethodsThe gene ontology (GO) annotationsComputational promoter analysis Materials and methodsPRIMA softwareQuantitative real-time RT-PCR Materials and methodsFive micrograms of total RNAcDNA oligo(dT) SuperScript II RNase H- reverse transcriptasereal-time PCR討論 RNAi and microarray technologies and a

12、 recently developed computational tool are powerful off-target effects computational promoter analysis was highly enriched for the binding signature of ATF2/ATF3/Jun結(jié)論 RNAi, microarrays and computational promoter analysis 對(duì)於 dissection of transcriptional networks 的研討是有力的 Targeting the primary activator of a DNA damage response network, the upstream regulator(ATM) was indeed required for