View
218
Download
0
Tags:
Embed Size (px)
Citation preview
2
前言• sequencing of the human -> functional gen
omics
• Gene-expression microarrays and RNA interferences (RNAi)
• ATM/NFκB 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
7• www.biocarta.com/ pathfi les/m_atmPathway.asp
ATM/p53 -mediated
ATM/NFkB -mediated
G1 checkpoint
Ataxia- telangiectasia ( AT)
8
www.mbb.yale.edu/ fl/fl_s_ghosh.htm
NFkB
ATM/NFkB -mediated
9• http://www.emdbiosciences.com/popup/cbc/NFKB_Interactive_Pathway.htm
ATM/NFkB -mediatedNFkB
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
Microarrayanalysis
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 analysisGO functional gene annotations
建立 siRNA knocked-downcellular systems
12
建立 siRNA knocked-downcellular systems
• Materials and methodsDNA 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。
14
Sample preparation and microarray hybridization
HEK293 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 : 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 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 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
17
Cluster analysis
• Materials and methods
112 gene 使用 the EXPANDER package 去做 average-linkagehierarchical 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
22
討論• RNAi and microarray technologies and a r
ecently 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 required for the induction of much of the network, the two downstream regulators (p53/NFkB)mediated the activation of largely disjoint sets of genes
• Statistical tests 聯合 computational promoter analysis 是高精確的