RNAi. Martin Latterich

RNAi

Contributors i x

Abbreviations ( xi

Preface xiii

1 Methods in RNA interference 1

Martin Latterich and Dalia Halawan i

References 2

2 RNAi reagent design 3

Bernd Jagla and Nathalie Aulne r

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 1 2

2 .5 .1 siRNA databases 1 2

2 .5 .2 miRNA databases 1 3

2 .6 Target RNA secondary structure predictions 1 3

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 .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 3 6

4 .3 .2 Detection of protein levels 3 7

4 .3 .3 Detection of knockdown efficiency using a reporter syste m

(surrogate assays) 38

4 .4 Silencing validation 38

4 .5 siRNA specificity 39

4 .6 Minimizing cell defense mechanism (dsRNA interferon response) 4 0

4 .7 Conclusion 4 1

Acknowledgments 42

References 4 2

5 RNAi libraries in dissecting molecular pathways of the human cell 4 7 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 5 0

5 .4 High-throughput RNAi screens 5 2

5 .5 RNAi-induced phenotype selections 5 4

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 _{6 6}

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 8 2

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 8 4

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 .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 11 9

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