1 Introduction Instructors and students introduce themselves (everyone will briefly talk about their background, and what their interests relevant to the course are).

Introduction to the course (30 min)
Common Literature Search Tools (30 min)
Description of Standard Molecular Techniques (30 min)
2 The Discovery of RNA Interference 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, 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, and E. Ullu. "Double-stranded RNA Induces mRNA Degradation in Trypanosoma Brucei." PNAS 95 (1998): 14687-14692.
3 The Basic Mechanism of RNAi 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, 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, and P. A. Sharp. "Targeted mRNA Degradation by Double-stranded RNA In Vitro." Genes & Dev 13 (1999): 3191-3197.
4 Physiological Relevance of the RNAi Pathway 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, 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, and D. P. Bartel. "MicroRNAs Modulate Hematopoietic Lineage Differentiation." Science 303 (2004): 83-86.
5 Short-interfering RNAs (siRNAs) and Their Delivery to Cells 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, 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, and T. Tuschl. "Duplexes of 21-nucleotide RNAs Mediate RNA Interference in Cultured Mammalian Cells." Nature 411 (2001): 494-498.
6 Stable Expression of siRNA: Short Hairpins and Expression Vectors 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, and R. Agami. "A System for Stable Expression of Short-interfering RNAs in Mammalian Cells." Science 296 (2002): 550-553.

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 Generation of Transgenic Animals for in vivo Silencing 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, 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, 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 Field Trip to Mouse-transgenesis Facility 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.  
9 Discussion of First written Assignment    
10 Design of Potent siRNA Sequences 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, 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, and A. Khvorova. "Rational siRNA Design for RNA Interference." Nature Biotech 22 (2004): 326-330.
11 Large-scale Genetic Analyses using RNAi 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, 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, and G. J. Hannon. "A Resource for Large-scale RNA-interference-based Screens in Mammals." Nature 428 (2004): 427-431.
12 Student Oral Presentations of Their Chosen Papers    
13 Using RNAi in the Prevention of Disease 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, 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, and J. Chen. "Inhibition of Influenza Virus Production in Virus-infected Mice by RNA Interference." PNAS 100 (2003): 2718-2723.
14 Course Summary and Discussion on the Future of RNAi 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, and I. M. Verma. "CRE Recombinase-inducible RNA Interference Mediated by Lentiviral Vectors." PNAS 101 (2004): 7347-7351.