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| 1 |
緒論
Introduction |
導師與學生自我介紹（每個人要簡要介紹自己的背景和他們的愛好與本課程的關係。）
Instructors and students introduce themselves (everyone will briefly talk about their background, and what their interests relevant to the course are).
課程介紹（30分鐘）
Introduction to the course (30 min)
普通文獻研究工具（30分鐘）
Common Literature Search Tools (30 min)
標準分子技術描述（30分鐘）
Description of Standard Molecular Techniques (30 min) |
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| 2 |
RNA干擾的發現
The Discovery of RNA Interference |
本課中，我們討論初步描述有關雙股RNA怎樣有效地讓基因表達關閉的論文
In this class, we will discuss the papers that first described how double-stranded RNA potently silences gene expression. |
Fire, A., S. Xu, M. K. Montgomery, S. A. Kostas, S. E. Driver和C. C. Mello.
〈雙股RNA在秀麗隱杆線蟲中有效和特殊的基因干擾〉，《自然》 391 (1998): 806-811
Fire, A., S. Xu, M. K. Montgomery, S. A. Kostas, S. E. Driver, and C. C. Mello. "Potent and Specific Genetic Interference by Double-stranded RNA in Caenorhabditis Elegans." Nature 391 (1998): 806-811.
Ngo, H., C. Tschudi, K. Gull和E. Ullu.
〈在布氏錐蟲中雙股RNA引導mRNA降解〉，《美國國家科學院院刊》95 (1998): 14687-14692
Ngo, H., C. Tschudi, K. Gull, and E. Ullu. "Double-stranded RNA Induces mRNA Degradation in Trypanosoma Brucei." PNAS 95 (1998): 14687-14692. |
| 3 |
RNAi的基本機制
The Basic Mechanism of RNAi |
以在班中討論的兩篇論文為開始研究RNAi的機制。第一組（Tabara等）揭露的被隨機突變捲入RNAi的基因。在另一篇論文中（Tuschl等），作者檢查在試管系統中估測基因關閉的動力學和RNAi的效率。
The two papers discussed in this class started to investigate the mechanism of RNA interference. The first group (Tabara et al.) uncovered genes involved in RNAi by random mutagenesis. In the other paper (Tuschl et al.), the authors examined gene silencing in an in vitro system to evaluate the kinetics and efficiency of RNAi. |
Tabara, H., M. Sarkissian, W. G. Kelly, J. Fleenor, A. Grishnok, L. Timmons, A. Fire和C. C. Mello. 〈秀麗隱杆線蟲中的rde-1基因，RNA干擾與轉位子關閉〉，《細胞》99 (1999): 123-132.
Tabara, H., M. Sarkissian, W. G. Kelly, J. Fleenor, A. Grishnok, L. Timmons, A. Fire, and C. C. Mello. "The rde-1 Gene, RNA Interference and Transposon Silencing in C. Elegans." Cell 99 (1999): 123-132.
Tuschl, T., P. D. Zamore, R. Lehmann, D. P. Bartel和P. A. Sharp. 〈體外雙股RNA標定的mRNA降解〉，《基因與發育》13 (1999): 3191-3197.
Tuschl, T., P. D. Zamore, R. Lehmann, D. P. Bartel, and P. A. Sharp. "Targeted mRNA Degradation by Double-stranded RNA In Vitro." Genes & Dev 13 (1999): 3191-3197. |
| 4 |
RNAi路徑之生理學關聯
Physiological Relevance of the RNAi Pathway |
在這課中，我們將要討論兩篇論文，分別是研究不為蛋白質編碼的（Reinhart等）或單個細胞類型（Chen等）的內源RNA分子是怎樣可以調節整個生物體的發展的。
In this session, we will discuss two papers that investigated how endogenous RNA molecules that do not code for protein can regulate the development of a whole organism (Reinhart et al.) or that of a single cell type (Chen et al.). |
Reinhart, B. J., F. J. Slack, M. Basson, A. E. Pasquinelli, J. C. Bettinger, A. E. Rougvie, H. R. Horvitz和G. Ruvkun.〈21核苷酸L7RNA在秀麗隱杆線蟲中調節發展的時機〉，《自然》403 (2000): 901-906.
Reinhart, B. J., F. J. Slack, M. Basson, A. E. Pasquinelli, J. C. Bettinger, A. E. Rougvie, H. R. Horvitz, and G. Ruvkun. "The 21-nucleotide let-7 RNA Regulates Developmental Timing in Caenorhabditis Elegans." Nature 403 (2000): 901-906.
Chen, C. Z., L. Li, H. F. Lodish和D. P. Bartel.〈微型RNA調節造血血統的區別〉，《科學》303 (2004): 83-86
Chen, C. Z., L. Li, H. F. Lodish, and D. P. Bartel. "MicroRNAs Modulate Hematopoietic Lineage Differentiation." Science 303 (2004): 83-86. |
| 5 |
短干擾RNA（siRNA）和他們的細胞傳遞
Short-interfering RNAs (siRNAs) and Their Delivery to Cells |
本課主題為兩份來自同一組的論文，最早描述RNAi的介體是RNA短鏈（約為21核苷酸，而且這些介體可以於不同哺乳動物內關閉基因的。
The subject of this class are two papers from the same group that first described that the mediators of RNAi were short (~21 nucleotide) fragments of RNA and that these could be used to silence genes in various mammalian cells. |
Elbashir, S. M., W. Lendeckel和T. Tuschl.〈以21或22核苷RNA為中介的RNA干擾〉，《基因與發育》15 (2001): 188-200.
Elbashir, S. M., W. Lendeckel, and T. Tuschl. "RNA Interference is Mediated by 21- and 22-nucleotide RNAs." Genes & Dev 15 (2001): 188-200.
Elbashir, S. M., J. Harborth, W. Lendeckel, A. Yalcin, K. Weber和T. Tuschl.〈培養哺乳動物細胞中以21核苷RNA為媒介的RNAi的雙方〉，《自然》411 (2001): 494-498.
Elbashir, S. M., J. Harborth, W. Lendeckel, A. Yalcin, K. Weber, and T. Tuschl. "Duplexes of 21-nucleotide RNAs Mediate RNA Interference in Cultured Mammalian Cells." Nature 411 (2001): 494-498. |
| 6 |
siRNA的穩定表達：短髮卡式連接與表達載體
Stable Expression of siRNA: Short Hairpins and Expression Vectors |
本課重點為siRNA的穩定表達。這是歷史上RNAi得以廣泛延伸於應用技術的各領域之重要臺階。這兩篇文章展示表達載體第一次用來推動外源siRNA在細胞內的表達。
This class will focus on the stable siRNA expression, which was a crucial step in the history of RNAi that widely expanded the realm of applications of this technology. The two papers that will be discussed showed for the first time that expression vectors could be used to drive the expression of exogenous siRNA inside cells. |
Brummelkamp, T., R. Bernards和R. Agami.〈siRNA在哺乳動物細胞中穩定表達的系統〉，《科學》Science 296 (2002): 550-553.
Brummelkamp, T., R. Bernards, and R. Agami. "A System for Stable Expression of Short-interfering RNAs in Mammalian Cells." Science 296 (2002): 550-553.
Miyagishi, M.和K. Taira.〈U6促進的四尿苷懸於3'端的siRNA有效抑制目標基因在哺乳動物細胞中的表達〉，《自然－生物技術》19 (2002): 497-500.
Miyagishi, M. and K. Taira. "U6 Promoter-driven siRNAs with Four Uridine 3' Overhangs Efficiently Suppress Targeted Gene Expression in Mammalian Cells." Nature Biotech 19 (2002): 497-500. |
| 7 |
基因轉殖動物在自然條件下的關閉的傳代
Generation of Transgenic Animals for in vivo Silencing |
緊跟siRNA可利用的表達載體能夠穩定地製造的論證，幾個團體製造了某個特殊基因被RNAi永久關閉的基因轉殖鼠。第一篇論文講述用病毒攜帶者傳遞所需DNA進入胚胎(Rubinson等)，並在動物成體中實現了永久性的基因關閉。第二篇提出了是否RNAi在不同基因背景下造成的關閉影響總是可預料及可再生的重要論點。這是一個探索RNAi是否可以用作更確定的而且漫長又艱辛的標定基因突變的本質問題。
Following the demonstration that siRNA could be stably produced using expression vectors, several groups generated transgenic mice in which a particular gene is permanently silenced by RNAi. The first paper used viral vectors to deliver the required DNA into embryos (Rubinson et al.), and achieved permanent gene silencing in adult animals. The second paper addressed the important issue of whether the effect of silencing by RNAi is always predictable and reproducible in different genetic backgrounds. This is an essential problem to investigate if RNAi is to be used as a substitute for the more established, albeit lengthy and difficult, targeted gene mutation approach. |
Rubinson, D. A., C. P. Dillon, A. V. Kwiatkowski, C. Sievers, L. Yang, J. Kopinja, M. Zhang, M. T. McManus, F. B. Gertler, M. L. Scott和L. van Parijs. 〈利用RNA干擾及基於水生環境病毒系統令哺乳動物主要細胞、幹細胞和基因轉殖鼠實現官能上的基因關閉〉，《自然－遺傳學》33 (2003): 401-407.
Rubinson, D. A., C. P. Dillon, A. V. Kwiatkowski, C. Sievers, L. Yang, J. Kopinja, M. Zhang, M. T. McManus, F. B. Gertler, M. L. Scott, and L. van Parijs. "A Lentivirus-based System to Functionally Silence Genes in Primary Mammalian Cells, Stem Cells and Transgenic Mice by RNA Interference." Nature Genetics 33 (2003): 401-407.
Simmer, F., M. Tijsterman, S. Parrish, S. P. Koushika, M. L. Nonet, A. Fire, J. Ahringer和R. H. A. Plasterk.〈假定定向的RNA聚合酶RRF3令秀麗隱杆線蟲對RNAi極為敏感的損失〉《當代生物學》12 (2002): 1317-1319.
Simmer, F., M. Tijsterman, S. Parrish, S. P. Koushika, M. L. Nonet, A. Fire, J. Ahringer, and R. H. A. Plasterk. "Loss of the Putative RNA-directed RNA Polymerase RRF3 Makes C. elegans Hypersensitive to RNAi." Current Biology 12 (2002): 1317-1319. |
| 8 |
鼠類基因轉殖研究所的實地考察
Field Trip to Mouse-transgenesis Facility |
我們將造訪用lentivirus繁殖“可拆開的”動物的基因轉殖鼠類研究所。我們會用通過此方法繁殖的活體基因轉殖鼠使綠色熒光蛋白表達形象化，並討論與鼠類基因轉殖有關的話題。
We will visit the mouse-transgenesis facility, where lentivirus is used to generate 'knock-down' animals. We will visualize GFP expression in live transgenic mice that have been generated by this method, and discuss issues relevant to mouse transgenesis. |
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| 9 |
對第一份書面作業的討論
Discussion of First written Assignment |
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| 10 |
siRNA有效序列的基因圖
Design of Potent siRNA Sequences |
以往siRNA序列的設計表現出較低效率，調查人在選擇有效序列時亦缺乏合理規律。兩團體最近更加系統地處理此問題。通過嚴格分析許多序列和他們關閉基因表達的相關能力，這些研究者測定出了設計有效siRNA序列的最重要因素。
The design of siRNA sequences previously appeared to be relatively inefficient, and investigators lacked rational rules for choosing effective sequences. Two groups recently approached this issue more systematically. By rigorous analysis of a large number of sequences and their relative abilities to silence gene expression, these researchers determined which factors were most important in the design of potent siRNA sequences. |
Schwarz, D. S., G. Hutvagner, T. Du, Z. Xu, N. Aronin和P. D. Zamore. "〈RNAi酶聯合體集合中的不對稱性〉，《細胞》115 (2003): 199-208.
Schwarz, D. S., G. Hutvagner, T. Du, Z. Xu, N. Aronin, and P. D. Zamore. "Asymmetry in the Assembly of the RNAi Enzyme Complex." Cell 115 (2003): 199-208.
Reynolds, A., D. Leake, Q. Boese, S. Scaringe, W. S. Marshall和A. Khvorova.〈為RNAi設計的siRNA的合理性〉,《自然－生物技術22 (2004): 326-330.
Reynolds, A., D. Leake, Q. Boese, S. Scaringe, W. S. Marshall, and A. Khvorova. "Rational siRNA Design for RNA Interference." Nature Biotech 22 (2004): 326-330. |
| 11 |
用RNAi進行大規模基因分析
Large-scale Genetic Analyses using RNAi |
如今siRNA的交遞方法和有效序列的設計已有很大的進步，一些團體已經開始用RNAi實現大規模的基因分析。這兩篇論文生成了通過高吞吐量的方式關閉大量的基因的siRNA庫。這樣的應用在未來將會更加普及，例如在藥物設計的定位發現組織中的顯著作用。
Now that delivery methods of siRNA and the design of effective sequences have greatly improved, several groups have started to carry out large-scale genetic analyses by RNAi. The two papers we will discuss both generated libraries of siRNA's to silence a vast number of genes in a high-throughput manner. Such applications should become more common in the future, particularly in a target-discovery context of drug-design, for example. |
Berns, K., E. M. Hijmans, J. Mullenders, T. R. Brummelkamp, A. Velds, M. Heimerikx, R. M. Kerkhoven, M. Madiredjo, W. Nijkamp, B. Weigelt, R. Agami, W. Ge, G. Cavet, P. S. Linsley, R. L. Beijersbergen和R. Bernards. 〈人類細胞中大規模RNAi屏蔽以識別p53路徑中的新成分〉， 《自然》 428 (2004): 431-437.
Berns, K., E. M. Hijmans, J. Mullenders, T. R. Brummelkamp, A. Velds, M. Heimerikx, R. M. Kerkhoven, M. Madiredjo, W. Nijkamp, B. Weigelt, R. Agami, W. Ge, G. Cavet, P. S. Linsley, R. L. Beijersbergen, and R. Bernards. "A Large-scale RNAi Screen in Human Cells Identifies New Components of the p53 Pathway." Nature 428 (2004): 431-437.
Paddison, P., J. M. Silva, D. S. Conklin, M. Schlaback, M. Li, S. Aruleba, V. Balija, A. O'Shaughnessy, L. Gnoj, K. Scobie, K. Chang, T. Westbrook, M. Cleary, R. Sachidanandam, W. R. McCombie, S. J. Elledge和G. J. Hannon.〈哺乳動物中基於RNAi的大規模屏蔽之方法〉，《自然》428 (2004): 427-431.
Paddison, P., J. M. Silva, D. S. Conklin, M. Schlaback, M. Li, S. Aruleba, V. Balija, A. O'Shaughnessy, L. Gnoj, K. Scobie, K. Chang, T. Westbrook, M. Cleary, R. Sachidanandam, W. R. McCombie, S. J. Elledge, and G. J. Hannon. "A Resource for Large-scale RNA-interference-based Screens in Mammals." Nature 428 (2004): 427-431. |
| 12 |
學生對所選論文的口頭介紹
Student Oral Presentations of Their Chosen Papers |
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| 13 |
疾病防治中的RNAi使用
Using RNAi in the Prevention of Disease |
除了做基礎研究的有力工具外，在臨床上使用RNAi關閉基因亦有很大希望。在此課中我們會討論嘗試利用RNAi治療疾病的兩個研究案例。第一篇中作者用向動物靜脈注射方法傳遞siRNA的方法防治急性肝炎(Song等)。第二個研究(Ge等)中，利用一個相似的處理對抗鼠群中流行性感冒的傳播。
In addition to being a powerful tool for basic research, the use of RNAi for the silencing of genes holds much promise in a clinical context. In this session, we will discuss two examples of studies that attempted to treat disease by RNAi. In the first paper, the authors prevented acute hepatitis by delivering siRNA to animals intravenously (Song et al.). In the second study (Ge et al.), a similar approach was used to fight influenza infection in mice. |
Song, E., S. K. Lee, J. Wang, N. Ince, N. Ouyang, J. Min, J. Chen, P. Shankar和J. Lieberman. 〈RNA干擾關注Fas在流行肝炎中對鼠類的保護〉，《自然醫學》9 (2003): 347-351.
Song, E., S. K. Lee, J. Wang, N. Ince, N. Ouyang, J. Min, J. Chen, P. Shankar, and J. Lieberman. "RNA Interference Targeting Fas Protects Mice from Fulminant Hepatitis." Nature Med. 9 (2003): 347-351.
Ge, Q., L. Filip, A. Bai, T. Nguyen, H. N. Eisen和J. Chen.〈通過RNAi阻止在已被病毒感染的鼠類中流感病毒的製造〉，《美國國家科學院院刊》100 (2003): 2718-2723.
Ge, Q., L. Filip, A. Bai, T. Nguyen, H. N. Eisen, and J. Chen. "Inhibition of Influenza Virus Production in Virus-infected Mice by RNA Interference." PNAS 100 (2003): 2718-2723. |
| 14 |
課程總結和對RNAi未來的討論
Course Summary and Discussion on the Future of RNAi |
最後一課中我們討論研究者現今是如何嘗試在特定組織或以誘導藥物的方式傳遞siRNA。我們概括此課程並思索siRNA的未來。（考慮到這是最後一堂討論課，所以僅一份論文。）
In the final class, we will discuss how researchers are now trying to deliver siRNA in a tissue specific or drug-inducible manner. We will sum up the course and speculate on the future of RNAi. (One paper only, to allow for final discussion of the course). |
Tiscornia, G., V. Tergaonkar, F. Galimi和I. M. Verma. 〈以水生環境病毒載體為媒介的CRE可誘導重組酶的RNA干擾〉，《美國國家科學院院刊》101 (2004): 7347-7351.
Tiscornia, G., V. Tergaonkar, F. Galimi, and I. M. Verma. "CRE Recombinase-inducible RNA Interference Mediated by Lentiviral Vectors." PNAS 101 (2004): 7347-7351. |
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