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Nobel Prize 2006

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  1. Nobel Prize 2006 RNA interference - gene silencing by double-stranded RNA

  2. 遺傳中心法則

  3. Andrew Z. Fire Craig C. Mello • 1986年始進行「線蟲早期發展的基因調控」的研究→和線蟲結下不解之緣 • 直至2003年,Fire,一直以線蟲為模型,從事遺傳變異影響細胞和生物體的相關研究。 • 04年獲選為美國科學院院士 • 1982年加入David Hirsh的實驗室並在那奠下分子生物學之基礎 • 契機:進入梅洛的實驗室,以線蟲作為模式生物,進行胚胎幹細胞分化和發育的研究。 • 2005年獲選為美國科學院院士。

  4. Pigmented petunia experiment,1990 • Chalcone synthase (CHS) • They attempted to overexpress chalcone synthase (CHS) • Unexpected results • Forty-two percent of plants presented abnormal. • Unclear mechanism

  5. Virus-induced recovery • N. clevelandii plant • Tomato black ring nepovirus strain W22 • Primary and secondary inoculation • Recovery

  6. Resistance in the recovered leaves is specific for viruses that have RNA sequences that are similar to the virus used for primary inoculation.

  7. 1.將unc-22 gene注入C. elegans

  8. 實驗前須知 • GFP :green fluorescent protein 綠色螢光蛋白,在螢光顯微鏡下可以觀察到螢光 • 實驗前:每個線蟲都被注入gfp-gene →意即:每隻線蟲在正常情況下都會轉譯出GFP蛋白,發出綠色螢光

  9. 實驗前須知 針對三種基因: • UNC-22gene:轉錄轉譯出肌絲蛋白,缺乏此蛋白則線蟲會抽搐 • GFP gene:轉錄轉譯出綠色螢光蛋白 • LacZL gene:一種在線蟲體內本來就不會轉錄轉譯出蛋白質的基因,作為對照組

  10. GFP篩選實驗 控制組: ds-Unc22RNA 應變組: ds-gfpG RNA 控制組: ds-lacZL RNA 幼蟲 成蟲成蟲體壁放大圖

  11. 胚胎觀察 對照組:未使用原位雜交 親代正常表現mex3之胚胎 親代生殖腺注入 antisense mex-3 RNA之胚胎 親代生殖腺注入 ds mex-3RNA之胚胎

  12. mRNA轉錄後的抑制 Antisense oligonucleotide Ribozyme RNAi miRNA dsRNA siRNA

  13. Ribozyme

  14. Antisense oligonucleotide

  15. Control of RNAi and gene expression 外源性dsRNA Affection of virus Transposons Exogenous RNA →siRNA 內源性dsRNA miRNA

  16. Dicer RNase III domains -cleave dsRNA or pre-miRNA connector helix -解dsRNA螺旋 platform domain -amino-terminal helicase domain -和RNase搭配作用 PAZ domain -附著dsRNA 3’

  17. RISC (RNA-induced silencing complex) PAZ domain -from dicer N domain -connect PAZ and PIWI PIWI domain - degrade mRNA Mid domain -穩定siRNA 5’ 及mRNA 3’

  18. RNA interference-siRNA Dicer cleaves dsRNA dsRNAis cleaved into small fragments siRNA binds with RISC mRNA is degraded

  19. miRNA

  20. c-Myc-regulated microRNAs • miR-17~miR-92

  21. RNAi信號擴增系統 • 利用被降解的mRNA,經過RdRp的作用形成新的dsRNA,使其進入siRNA的RNAi系統,達到信號擴增的效果

  22. 信號擴增系統動畫

  23. RNAi application • The source of siRNA: 1.synthesize in test tube PCR siRNA → target cells 2.synthesize in organism siRNA → target cells(replication)

  24. 生物體內原本就有的基因序列,選取其中的一小部分生物體內原本就有的基因序列,選取其中的一小部分 轉錄此股,則可形成所需的hairpin siRNA Design a siRNA expression vector

  25. Design a siRNA sequence • Choose proper siRNA expression vector • 5~6 A(T) tail • Select different parts of sequence of several target genes • Ex: AU···CCGA···AGCCUAC···UCCUUAU

  26. RNAi生醫應用實例 • 2003年美國Acuity藥廠開始應用RNAi方法治療macular degeneration,初步有良好效果。 • Sirna治療學公司宣佈他們實現了RNAi應用於治療中的重大突破,成功抑制了HBV病毒在人體內的95%的表現。 • 哈佛醫學院的研究人員證明利用RNAi技術能夠抑制LDH-A,並因此將癌細胞的生長率降低100倍。

  27. RNAi vs. HIV • 據科學家報導,他們設計了一種新的愛滋病治療方法,用抗體攜帶RNAi直接傳遞給機體免疫細胞

  28. 缺點 • 抑制受體可能對細胞有不良影響 • HIV reversetranscriptase突變 • HIV 基因變異大,使設計siRNA困難 • 抗體-siRNA複合物可能對免疫系統刺激

  29. Reference • http://www.ambion.com/techlib/tb/tb_506.html (siRNA Design Guidelines) • http://www.genscript.com/siRNA_technology.html (siRNA Technology) • http://www.ambion.com/techlib/tn/95/952.html (siRNA載體) • http://content.nejm.org/cgi/content/full/347/17/1364(RNAi應用) • http://www.nature.com/nature/journal/v391/n6669/pdf/391806a0.pdf (Nature journal)

  30. Reference • http://rnajournal.cshlp.org/content/12/7/1161.full.pdf (Post-transcriptional regulation of microRNA expression) • http://www.landesbioscience.com/journals/cbt/article/HanCBT6-8.pdf (DNA Methylation Regulates MicroRNA Expression) • http://nobelprize.org/nobel_prizes/medicine/laureates/2006/index.html (Nobel Prize2006) • http://en.wikipedia.org/wiki/C_elegans (C.elegans from wikipedia) • http://upload.wikimedia.org/wikipedia/commons/b/be/CrawlingCelegans.gif (photo from wikipedia)

  31. Reference • http://nobelprize.org/nobel_prizes/medicine/laureates/2006/fire-autobio.html(費爾自傳) • http://www.biox.cn/content/200610/20061002180126_322511.shtml (2006年诺贝尔医学奖获得者--生命经纬) • http://nobelprize.org/nobel_prizes/medicine/laureates/2006/mello-autobio.html(枚落自傳) • http://web1.nsc.gov.tw/ct.aspx?xItem=8539&ctNode=40&mp=1 (核醣核酸干擾--不容忽視的沉默) • http://www.newsoo.cn/epaper/yyjjb/2007/05/18/484274.htm (被RNA“干扰”的人)

  32. Reference • http://www.plantcell.org/cgi/reprint/2/4/279.pdf (Introduction of a Chimeric Chalcone Synthase Gene into Petunia Results in Reversible Co-Suppression of Homologous Genes in trans) • http://www.sciencemag.org/cgi/content/full/276/5318/1558 (A Similarity Between Viral Defense and Gene Silencing in Plants • http://porpax.bio.miami.edu/~cmallery/255/255life/ribozyme.jpg)(ribozyme 圖)  • http://www.genscript.com/images/sirna/miRNA.jpg (miRNA機制圖)

  33. Reference • 維基百科http://en.wikipedia.org/wiki/Dicerhttp://en.wikipedia.org/wiki/RNA_interferencehttp://en.wikipedia.org/wiki/RNA-induced_silencing_complex • 某人的部落格http://huiweno824.spaces.live.com/blog/cns!92AA8BE887664BCD!330.entry • 某醫學中心http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=15845026http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1370384

  34. Reference • http://www.afv.org.hk/AMD_c.htm(年齡相關性黃斑點退化) • http://www.ebiotrade.com/newsf/2008-8/200888154931.htm(生物通) • http://content.nejm.org/cgi/content/full/347/17/1364/F2(The NEW ENGLAND JOURNAL of MEDICINE) • http://www.medscape.com/pi/editorial/cmecircle/2000/117/baldassano1/slide27.gif