RNAi Martin Latterich
Contributors Abbreviations Preface i x ( xi xiii 1 Methods in RNA interference 1 Martin Latterich and Dalia Halawan i References 2 2 RNAi reagent design 3 Bernd Jagla and Nathalie Aulner 2.1 Introduction 3 2.2 Lessons learned from X-ray structures/mechanism 4 2.3 Current design considerations 5 2.3.1 Asymmetry 5 2.3.2 Sequence positional preferences 6 2.3.3 Practical features 6 2.3.4 Non-sequence position-based considerations 6 2.3.5 shrna design considerations 8 2.4 Prediction tools 9 2.4.1 Other related tools 1 1 2.5 Databases 12 2.5.1 sirna databases 1 2 2.5.2 mirna databases 1 3 2.6 Target RNA secondary structure predictions 13 2.7 Other resources on the web 1 5 2.8 Concluding remarks 1 5 Acknowledgments 1 6 References 1 6 3 RNAi - a chemical perspective 2 1 Ouathek Ouerfell i 3.1 Background 2 1 3.2 Introduction 22 3.3 sirnas versus shrnas 22 3.3.1 sirnas 23 3.3.2 shrnas 23 3.4 RNAi reagents 23 3.5 RNA chemistry 24
3.6 RNA synthesis methods 2 4 3.7 High-throughput sirna synthesis: the full process 2 6 3.8 Summary and outlook 2 7 Acknowledgments 2 8 References 28 4 Validation of RNAi 3 1 Nathalie Aulner and Bernd Jagla 4.1 Introduction 3 1 4.2 sirna delivery 3 2 4.2.1 sirna transfection 3 2 4.2.2 Introduction of shrnas into mammalian cells 3 3 4.2.3 RNAi screening delivery systems 3 4 4.3 Silencing efficacy (potency) 3 5 4.3.1 Detection of mrna levels 36 4.3.2 Detection of protein levels 3 7 4.3.3 Detection of knockdown efficiency using a reporter system (surrogate assays) 38 4.4 Silencing validation 38 4.5 sirna specificity 39 4.6 Minimizing cell defense mechanism (dsrna interferon response) 40 4.7 Conclusion 4 1 Acknowledgments 42 References 4 2 5 RNAi libraries in dissecting molecular pathways of the human cell 47 Cheryl Eifert, Antonis Kourtidis and Douglas S. Conkli n 5.1 Introduction 4 7 5.2 RNAi 4 7 5.3 Approaches for loss-of-function screens 50 5.4 High-throughput RNAi screens 5 2 5.5 RNAi-induced phenotype selections 54 5.6 Screens for mirna functions 5 6 5.7 Perspectives in disease treatment 5 7 References 5 7 6 High-throughput RNAi in Caenorhabditis elegans - from molecular phenotypes to pathway analysis 6 5 Sarah Jenna and Eric Cheve t 6.1 Introduction 6 5 6.1.1 RNAi in C. elegans 6 5 6.1.2. High-throughput RNAi in C. elegans 6 6 6.2 The experiments 66 6.3 Summary 68 References 69 Protocol 6.1 : Generation of constructs driving RNAi through a feeding procedure 7 1 Protocol 6.2: RNAi treatment of GFP reporter animals 73 Protocol 6.3 : Sorting of fluorescent animals and measurement of the UPR 77
7 RNAi in Xenopus laevis 79 Adrianna L. Stromme and Craig A. Mandato 7.1 Introduction 79 7.2 Oocyte isolation 8 1 7.2.1 Inducing ovulation 8 1 7.2.2 Collecting eggs 8 1 7.3 Testes isolation 82 7.4 In vitro fertilization 8 2 7.5 Microinjecting dsrna into embryos/oocytes 82 7.5.1 Dejellying embryos 8 2 7.5.2 Vitelline membrane removal 8 3 7.5.3 Microinjections 8 3 7.6 Lineage labeling 8 4 7.6.1 Dextran amines 8 4 7.6.2 ß-Galactosidase RNA 8 4 7.6.3 GFP RNA as a lineage marker 84 7.7 Screening of phenotypes 8 5 References 8 5 Protocol 7.1 : Solutions appendix 8 6 Protocol 7.2: X-gal staining protocol (Sive et al., 1997) 8 8 Protocol 7.3: Overall protocol for sirna experiment (example) 8 9 8 Generation of transgenic and knockdown mice with lentiviral vectors and RNAi techniques 9 1 Jenni Huusko, Petri I. Mäkinen, Leena Alhonen and Seppo Ylä-Herttual a 8.1 Introduction 9 1 8.2 Production of transgenic and knockdown mice 9 1 8.3 Use of ES cells 9 1 8.4 Use of embryos 9 2 8.5 Lentivirus vectors 9 3 8.6 Design of LVs for the generation of knockdown mice 9 4 8.6.1 Constitutive pol III promoters 9 4 8.6.2 Regulatable pol III promoters 9 5 8.6.3 Pol II promoters 9 5 References 9 6 Protocol 8.1: Mice, reagents and equipment 9 9 Protocol 8.2: Setting up capillaries, injection needles, injection chambers an d preparations for transgenesis 10 2 Protocol 8.3: Direct microinjection of the viral construct to the subzonal spac e (= perivitelline space) of a fertilized egg cell 10 5 Protocol 8.4: Zona pellucida removal and lentiviral transduction 10 8 9 RNAi in fungi 11 3 Hitoshi Nakayashiki 9.1 Introduction 11 3 9.1.1 The discovery of quelling in Neurospora 11 3 9.1.2 Meiotic silencing by unpaired DNA (MSUD), a nove l gene-silencing phenomenon in Neurospora 11 3 9.1.3 RNAi as a genetic tool in fungi 114
9.2 RNAi strategies in fungi 11 6 9.2.1 RNAi using a hairpin RNA-expressing plasmid 11 6 9.2.2 RNAi using an opposing-dual promoter system 11 7 9.2.3 Direct delivery of dsrna into fungal cells 11 8 9.2.4 Simultaneous silencing of multiple genes 11 8 9.3 Genetic transformation and RNAi protocols for fungi 119 Acknowledgments 12 0 References 120 Protocol 9.1 : Transformation of Magnaporthe oryzae by the calcium chloride/polyethylene glycol (PEG) method 12 3 Protocol 9.2: Transformation of Cryptococcus neoformans by electroporation 12 5 Protocol 9.3: Transformation of Mortierella alpina by the microparticl e bombardment method 12 7 Protocol 9.4: Transformation of Phytophthora infestans by th e Lipofectin-mediated transfection method 12 9 Index 133