Contributors ix
Abbreviations xi
Preface xiii
1 Methods in RNA interference 1
Martin Latterich and Dalia Halawani
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 11
2.5 Databases 12
2.5.1 siRNA databases 12
2.5.2 miRNA databases 13
2.6 Target RNA secondary structure predictions 13
2.7 Other resources on the web 15
2.8 Concluding remarks 15
Acknowledgments 16
References 16
3 RNAi – a chemical perspective 21
Ouathek Ouerfelli
3.1 Background 21
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 24
3.7 High-throughput siRNA synthesis: the full process 26
3.8 Summary and outlook 27
Acknowledgments 28
References 28
4 Validation of RNAi 31
Nathalie Aulner and Bernd Jagla
4.1 Introduction 31
4.2 siRNA delivery 32
4.2.1 siRNA transfection 32
4.2.2 Introduction of shRNAs into mammalian cells 33
4.2.3 RNAi screening delivery systems 34
4.3 Silencing efficacy (potency) 35
4.3.1 Detection of mRNA levels 36
4.3.2 Detection of protein levels 37
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 41
Acknowledgments 42
References 42
5 RNAi libraries in dissecting molecular pathways of the human cell 47
Cheryl Eifert, Antonis Kourtidis and Douglas S. Conklin
5.1 Introduction 47
5.2 RNAi 47
5.3 Approaches for loss-of-function screens 50
5.4 High-throughput RNAi screens 52
5.5 RNAi-induced phenotype selections 54
5.6 Screens for miRNA functions 56
5.7 Perspectives in disease treatment 57
References 57
6 High-throughput RNAi in Caenorhabditis elegans – from molecular
phenotypes to pathway analysis 65
Sarah Jenna and Eric Chevet
6.1 Introduction 65
6.1.1 RNAi in C. elegans 65
6.1.2. High-throughput RNAi in C. elegans 66
6.2 The experiments 66
6.3 Summary 68
References 69
Protocol 6.1: Generation of constructs driving RNAi through a feeding procedure 71
Protocol 6.2: RNAi treatment of GFP reporter animals 73
Protocol 6.3: Sorting of fluorescent animals and measurement of the UPR 77
vi Contents
7 RNAi in Xenopus laevis 79
Adrianna L. Stromme and Craig A. Mandato
7.1 Introduction 79
7.2 Oocyte isolation 81
7.2.1 Inducing ovulation 81
7.2.2 Collecting eggs 81
7.3 Testes isolation 82
7.4 In vitro fertilization 82
7.5 Microinjecting dsRNA into embryos/oocytes 82
7.5.1 Dejellying embryos 82
7.5.2 Vitelline membrane removal 83
7.5.3 Microinjections 83
7.6 Lineage labeling 84
7.6.1 Dextran amines 84
7.6.2 β-Galactosidase RNA 84
7.6.3 GFP RNA as a lineage marker 84
7.7 Screening of phenotypes 85
References 85
Protocol 7.1: Solutions appendix 86
Protocol 7.2: X-gal staining protocol (Sive et al., 1997) 88
Protocol 7.3: Overall protocol for siRNA experiment (example) 89
8 Generation of transgenic and knockdown mice with lentiviral vectors
and RNAi techniques 91
Jenni Huusko, Petri I. Mäkinen, Leena Alhonen and Seppo Ylä-Herttuala
8.1 Introduction 91
8.2 Production of transgenic and knockdown mice 91
8.3 Use of ES cells 91
8.4 Use of embryos 92
8.5 Lentivirus vectors 93
8.6 Design of LVs for the generation of knockdown mice 94
8.6.1 Constitutive pol III promoters 94
8.6.2 Regulatable pol III promoters 95
8.6.3 Pol II promoters 95
References 96
Protocol 8.1: Mice, reagents and equipment 99
Protocol 8.2: Setting up capillaries, injection needles, injection chambers and
preparations for transgenesis 102
Protocol 8.3: Direct microinjection of the viral construct to the subzonal space
(= perivitelline space) of a fertilized egg cell 105
Protocol 8.4: Zona pellucida removal and lentiviral transduction 108
9 RNAi in fungi 113
Hitoshi Nakayashiki
9.1 Introduction 113
9.1.1 The discovery of quelling in Neurospora 113
9.1.2 Meiotic silencing by unpaired DNA (MSUD), a novel
gene-silencing phenomenon in Neurospora 113
9.1.3 RNAi as a genetic tool in fungi 114
Contents vii
9.2 RNAi strategies in fungi 116
9.2.1 RNAi using a hairpin RNA-expressing plasmid 116
9.2.2 RNAi using an opposing-dual promoter system 117
9.2.3 Direct delivery of dsRNA into fungal cells 118
9.2.4 Simultaneous silencing of multiple genes 118
9.3 Genetic transformation and RNAi protocols for fungi 119
Acknowledgments 120
References 120
Protocol 9.1: Transformation of Magnaporthe oryzae by the calcium
chloride/polyethylene glycol (PEG) method 123
Protocol 9.2: Transformation of Cryptococcus neoformans by electroporation 125
Protocol 9.3: Transformation of Mortierella alpina by the microparticle
bombardment method 127
Protocol 9.4: Transformation of Phytophthora infestans by the
Lipofectin-mediated transfection method 129
Index 133
