课件生科4甲蔡德峰.ppt
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1、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 processe
2、s during normal development and in response to homeostatic challenges,4,Gene-expression microarrays,https:/www.vbi.vt.edu/ article/articleview/145,5,RNA interferences (RNAi),www.mpg.de/./ EEB/200432_035.shtml,(RNA-induced silencing complex),6,RNA interferences (RNAi),www.life.uiuc.edu/ shapiro/RNAiA
3、pps.html,7, pathfiles/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:/ -mediated,NFkB,ATM,10,Hypothesis,the combined experimental strategy of expression arrays and RNAi is indeed a
4、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 gene
5、s,*,*,*,*,*,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 fragm
6、ents,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 prep
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