RNAi. Martin Latterich

Transcription

1 RNAi Martin Latterich

2

3 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 Lessons learned from X-ray structures/mechanism Current design considerations Asymmetry Sequence positional preferences Practical features Non-sequence position-based considerations shrna design considerations Prediction tools Other related tools Databases sirna databases mirna databases Target RNA secondary structure predictions Other resources on the web Concluding remarks 1 5 Acknowledgments 1 6 References RNAi - a chemical perspective 2 1 Ouathek Ouerfell i 3.1 Background Introduction sirnas versus shrnas sirnas shrnas RNAi reagents RNA chemistry 24

4 3.6 RNA synthesis methods High-throughput sirna synthesis: the full process Summary and outlook 2 7 Acknowledgments 2 8 References 28 4 Validation of RNAi 3 1 Nathalie Aulner and Bernd Jagla 4.1 Introduction sirna delivery sirna transfection Introduction of shrnas into mammalian cells RNAi screening delivery systems Silencing efficacy (potency) Detection of mrna levels Detection of protein levels Detection of knockdown efficiency using a reporter system (surrogate assays) Silencing validation sirna specificity Minimizing cell defense mechanism (dsrna interferon response) Conclusion 4 1 Acknowledgments 42 References RNAi libraries in dissecting molecular pathways of the human cell 47 Cheryl Eifert, Antonis Kourtidis and Douglas S. Conkli n 5.1 Introduction RNAi Approaches for loss-of-function screens High-throughput RNAi screens RNAi-induced phenotype selections Screens for mirna functions Perspectives in disease treatment 5 7 References High-throughput RNAi in Caenorhabditis elegans - from molecular phenotypes to pathway analysis 6 5 Sarah Jenna and Eric Cheve t 6.1 Introduction RNAi in C. elegans High-throughput RNAi in C. elegans The experiments 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

5 7 RNAi in Xenopus laevis 79 Adrianna L. Stromme and Craig A. Mandato 7.1 Introduction Oocyte isolation Inducing ovulation Collecting eggs Testes isolation In vitro fertilization Microinjecting dsrna into embryos/oocytes Dejellying embryos Vitelline membrane removal Microinjections Lineage labeling Dextran amines ß-Galactosidase RNA GFP RNA as a lineage marker 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) 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 Production of transgenic and knockdown mice Use of ES cells Use of embryos Lentivirus vectors Design of LVs for the generation of knockdown mice Constitutive pol III promoters Regulatable pol III promoters 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 RNAi in fungi 11 3 Hitoshi Nakayashiki 9.1 Introduction The discovery of quelling in Neurospora Meiotic silencing by unpaired DNA (MSUD), a nove l gene-silencing phenomenon in Neurospora RNAi as a genetic tool in fungi 114

6 9.2 RNAi strategies in fungi RNAi using a hairpin RNA-expressing plasmid RNAi using an opposing-dual promoter system Direct delivery of dsrna into fungal cells Simultaneous silencing of multiple genes 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