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Page 1: Nobel Prize 2006

Nobel Prize 2006

RNA interference - gene silencing by double-stranded RNA

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遺傳中心法則

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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。

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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

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Virus-induced recovery

• N. clevelandii plant• Tomato black ring

nepovirus strain W22 • Primary and secondary

inoculation• Recovery

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

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1. 將 unc-22 gene注入 C. elegans

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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

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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

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GFP篩選實驗

幼蟲 成蟲成蟲體壁放大

控制組:ds-Unc22RNA

控制組:ds-lacZL RNA

應變組:ds-gfpG RNA

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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

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mRNA轉錄後的抑制•Antisense oligonucleotide•Ribozyme•RNAi

–miRNA–dsRNA–siRNA

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Ribozyme

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Antisense oligonucleotide

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Control of RNAi and gene expression

1.外源性 dsRNA – Affection of virus– Transposons– Exogenous RNA

→siRNA2.內源性 dsRNA

– miRNA

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DicerRNase III domains -cleave dsRNA or pre-miRNAconnector helix -解 dsRNA螺旋platform domain -amino-terminal helicase domain -和 RNase搭配作用PAZ domain -附著 dsRNA 3’

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RISC (RNA-induced silencing complex)

PAZ domain-from dicerN domain-connect PAZ and PIWI

PIWI domain- degrade mRNA

Mid domain-穩定 siRNA 5’ 及 mRNA 3’

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siRNA binds with RISC

Dicer cleaves dsRNA

RNA interference-siRNA

dsRNA is cleaved into small fragments

mRNA is degraded

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miRNA

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c-Myc-regulated microRNAs

• miR-17~miR-92

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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

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信號擴增系統動畫

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RNAi application

• The source of siRNA :1.synthesize in test tube PCR

siRNA → target cells2.synthesize in organism

siRNA → target cells ( replication )

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Design a siRNA expression vector

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

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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

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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。

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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

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缺點• 抑制受體可能對細胞有不良影響• HIV reverse transcriptase 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

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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)

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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)

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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 )

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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 )

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Reference

• 維基百科http://en.wikipedia.org/wiki/Dicerhttp://en.wikipedia.org/wiki/RNA_interference http://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=15845026 http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1370384

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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


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