Frontiers in RNAi

Volume 1

by

Ralph A. Tripp, Jon M. Karpilow

DOI: 10.2174/97816080594091140101
eISBN: 978-1-60805-940-9, 2014
ISBN: 978-1-60805-941-6
ISSN: 2352-8400 (Print)



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RNA Interference (RNAi) arrived in the scientific community just over a decade ago, and has since blossomed with new connections being...[view complete introduction]
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Table of Contents

Foreword

- Pp. i

Devin Leake

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Preface

- Pp. iii

Ralph Tripp and Jon Karpilow

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List of Contributors

- Pp. v-vii (3)

Ralph Tripp and Jon Karpilow

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Acknowledgments

- Pp. ix

Ralph Tripp and Jon Karpilow

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RNAi and Off-Target Effects

- Pp. 3-20 (18)

Amanda Birmingham, Andreas Kaufmann and Karol Kozak

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Automation Considerations for RNAi Library Formatting and High Throughput Transfection

- Pp. 21-39 (19)

Sean M. Johnston, Caroline E. Shamu and Jennifer A. Smith

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Public Repositories for RNAi Screening Data

- Pp. 40-57 (18)

Esther E. Schmidt, Michael S. Banos, Jennifer A. Smith, Amanda Birmingham, Michael Boutros and Caroline E. Shamu

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Pooled shRNA Screening

- Pp. 58-78 (21)

Annaleen Vermeulen, Anja van Brabant Smith, Sarah B. Anderson, Roderick L. Beijersbergen and Kaylene J. Simpson

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RNAi for Viral Disease Control

- Pp. 79-106 (28)

Cameron R. Stewart, S. Mark Tompkins, Kristie A. Jenkins, Leonard H. Izzard, John Stambas, Andrew G. Bean, Mark L. Tizard, Timothy J. Doran and John W. Lowenthal

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Host-Encoded miRNAs Involved in Host-Pathogen Interactions

- Pp. 107-143 (37)

Samantha Barichievy and Abhijeet Bakre

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RNAi Screening in Cells of the Immune System: Challenges and Opportunities

- Pp. 144-177 (34)

Sinu P. John, Michael Freeley, Aideen Long and Iain D.C. Fraser

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siRNA Microarray-Based Genomic Screening

- Pp. 178-214 (37)

Yong-Jun Kwon, HiChul Kim, Jin Y. Kim, Namyoul Kim, Jinyeoung Heo, TaeKyu Lee, Michael A.E. Hansen and Veronica Soloveva

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A Three-Dimensional Spheroid Cell Culture Model for Robust High-Throughput RNA Interference Screens

- Pp. 215-231 (17)

Geoffrey Bartholomeusz and Arvind Rao

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The Use of RNAi Technology in the Development of High Performance Bioproduction Cell Lines

- Pp. 232-246 (15)

Weilin Wu, Sabine van der Sanden, Paula Brooks, Jon M. Karpilow, Steven Oberste and Ralph A. Tripp

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RNAi Screening to Facilitate Drug Repurposing

- Pp. 247-265 (19)

Olivia Perwitasari and Ralph A. Tripp

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Author Index

- Pp. 266

Ralph A. Tripp and Jon M. Karpilow

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Subject Index

- Pp. 267-268 (2)

Ralph A. Tripp and Jon M. Karpilow

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Foreword

Great technologies follow similar roads to maturity. A breakthrough discovery leads to rapid early adoption which (more often than not) precedes the identification of limitations. Eventually, solutions are identified and the technology, with all its strengths and weaknesses, is put to the test through wide-spread implementation. This process of maturation takes years and in this regard, RNA Interference (RNAi) is no different than yeast two-hybrid, chip-based gene expression profiling, and monoclonal antibodies.

Once the scientific community overcame the sense of excitement associated with the discovery of non-coding RNA-based posttranscriptional gene regulation, early adopters between 2000-2005 immediately gained insights into the technology’s limitations. Not all siRNAs silenced with equal efficiency. Some siRNA designs activated the innate immune response. Other siRNAs exhibited off-target effects and could induce false positive phenotypes. To some, these challenges might have appeared overly daunting, but given the potential of RNAi, researchers in both academic and industrial settings pressed to find solutions. Algorithms were designed to address issues associated with functionality and position specific chemical modifications were adopted to minimize offtarget effects and activation of the innate immune system. Self-delivering siRNA were developed to address the need to deliver reagents to cell lines that were refractory to lipid-mediated delivery and viral-based gene silencing was constructed to facilitate experiments in systems that required extended periods of knockdown.

While challenges still persist (e.g., therapeutic delivery), overcoming the first wave of technical hurdles has been paramount to expansion of RNAi into new fields of interest. As evidenced in this ebook, continued mechanistic studies are now combined with a host of new developments where RNAi technology is being merged with miniaturized screening platforms, field-specific database infrastructures, host-pathogen interaction mapping, and new (3D) tissue culture models. At the same time, the technology is reaching into more applied fields. RNAi screening is slowly becoming a module of synthetic biology and cell line engineering, and a key component of therapeutic drug repurposing. This expansion of the technology mirrors the growth and development observed with other platforms (e.g., NG Sequencing, PCR) and is indicative that over the course of little more than a decade, RNAi has grown up!

Devin Leake
Vice President of Research and Development
Gen9 Inc.
Cambridge, MA
USA

Preface

RNA interference (RNAi), has developed into an important tool for gene function elucidation by leveraging this endogenous gene-silencing pathway with well-designed triggers for the systematic silencing of particular genes. Incorporation of RNAi into high throughput screening platforms has led to important discoveries in fields such as cancer biology and pathogenesis, successes driven by establishment of best-practices and developments in instrumentation, analysis, and the growing sophistication of cell-based assays. The following chapters represent some of the most important considerations and recent developments in RNAi screening technology. It is certainly our hope that this compendium contains something for everyone, both novice and experienced researchers alike.

The topics in this collection range from recent automation platforms for ultra-high throughput screens (see Chapters 2 and 8) to novel applications of this gene modulation technology to cells grown in 3D culture (see Chapter 9). Chapter 1 provides a review of RNAi as a research tool as well as experimental and bioinformatics approaches to reduce and identify off-target effects in highthroughput screening data. Important considerations regarding deposition of large data sets in public repositories are presented in Chapter 3. Pooled shRNA screening methods are discussed in Chapter 4. Chapters 5 and 6 describe how RNAi technologies are being used to interrogate the host-pathogen interface. RNAi screening in difficult-to-transfect immune cells is addressed in Chapter 7 and applications beyond target discovery are presented in Chapters 10 and 11.

While it is not evident at first glance, a research alliance underlies all the contributions detailed in this book. All authors are affiliated with a lab that participates in the RNAi Global Initiative, a world-wide association of biomedical researchers established to broaden and accelerate the utility and application of RNAi technology. Founded in 2005 with eleven member institutions, the organization focused on developing and disseminating information on the basics of RNAi screening. As the organization grew and technology developed, focus shifted toward more complex, often biological, challenges.

Nine years after its inauguration, the RNAi Global Initiative now includes over 60 academic institutes spread across five continents. Members continue to share their findings in a range of fields (stem cell biology, cancer, and host-pathogen interactions) and as evidenced by this book, collaborate in the development of new bioinfomatic tools, screening methods, and programs in applied biology. The longevity of this alliance is the result of continued contributions on the part of its membership and is a testament to the importance of collaboration in building a cohesive scientific community.

Ralph Tripp
University of Georgia
College of Veterinary Medicine
Department of Infectious Diseases
Athens, GA, USA

&

Jon Karpilow
Thermo Fisher Scientific
Lafayette, CO, USA

List of Contributors

Editor(s):
Ralph A. Tripp
Department of Infectious Diseases
University of Georgia
Athens
GA
USA


Jon M. Karpilow
Biosciences Division
Thermo Fisher Scientific
Lafayette
CO
USA




Contributor(s):
Abhijeet Bakre
Department of Infectious Diseases
University of Georgia, College of Veterinary Medicine
111 Carlton Street
Athens
GA 30602
USA


Aideen Long
Department of Clinical Medicine
Trinity College Dublin
Dublin
Ireland


Amanda Birmingham
Dharmacon
part of GE Healthcare
2650 Crescent Dr., Suite, 100
Lafayette
CO 80026
USA


Andreas Kaufmann
LMSC
ETH
Schafmattstrasse 18
8093 Zurich
Switzerland


Andrew G. Bean
CSIRO Australian Animal Health Laboratory
Geelong 3220
Victoria
Australia


Anja Van Brabant Smith
Dharmacon
part of GE Healthcare
2650 Crescent Dr., Suite, 100
Lafayette
CO 80026
USA


Annaleen Vermeulen
Dharmacon
part of GE Healthcare
2650 Crescent Dr., Suite, 100
Lafayette
CO 80026
USA


Arvind Rao
Department of Bioinformatics and Computational Biology
Division of Quantitative Sciences The University of Texas M.D
Anderson Cancer Center
Houston
TX 77054
USA


Cameron R. Stewart
CSIRO Australian Animal Health Laboratory
Geelong 3220
Victoria
Australia


Caroline E. Shamu
ICCB-Longwood Screening Facility
Harvard Medical School
250 Longwood Avenue, SGM Building Room 604
Boston
MA 021155731
USA


Esther E. Schmidt
German Cancer Research Center, Division Signaling and Functional Genomics and Heidelberg University
Department for Cell and Molecular Biology, Medical Faculty Mannheim
Im Neuenheimer Feld 580
69120 Heidelberg
Germany


Geoffrey Bartholomeusz
Department of Experimental Therapeutics
Division of Cancer Medicine, The University of Texas M.D. Anderson Cancer Center
Houston
TX. 77054
USA


HiChul Kim

Institute Pasteur Korea
696 Sampyeong-dong
Bundang-gu
Gyeonggi-do, 463-400
South Korea


Iain D.C. Fraser
Signaling Systems Unit, Laboratory of Systems Biology
National Institute of Allergy and Infectious Diseases
National Institutes of Health
Bethesda
MD 20892
USA


Jennifer A. Smith
ICCB-Longwood Screening Facility
Harvard Medical School, 250 Longwood Avenue
SGM Building Room 604
Boston
MA 021155731
USA


Jin Y. Kim
Institute Pasteur Korea
696 Sampyeong-dong
Bundang-gu
Gyeonggi-do, 463-400
South Korea


Jinyeoung Heo
Institute Pasteur Korea
696 Sampyeong-dong
Bundang-gu
Gyeonggi-do, 463-400
South Korea


John Stambas
Deakin University School of Medicine
Waurn Ponds
Victoria, 3216
Australia


John W. Lowenthal
CSIRO Australian Animal Health Laboratory
Geelong 3220
Victoria
Australia


Jon M. Karpilow
Thermo Fisher Scientific
2650 Crescent Dr., Suite, 202
Lafayette
CO 80026
USA


Karol Kozak
LMSC
ETH
Schafmattstrasse 18
8093 Zurich
Switzerland


Kaylene J. Simpson
Victorian Centre for Functional Genomics
Peter MacCallum Cancer Centre
East Melbourne
Victoria, 3002
Australia


Kristie A. Jenkins
CSIRO Australian Animal Health Laboratory
Geelong 3220
Victoria
Australia


Leonard H. Izzard
Deakin University School of Medicine
Waurn Ponds
Victoria, 3216
Australia


Mark L. Tizard
CSIRO Australian Animal Health Laboratory
Geelong 3220
Victoria
Australia


Michael A. E. Hansen
Institute Pasteur Korea
696 Sampyeong-dong
Bundang-gu
Gyeonggi-do, 463-400
South Korea


Michael Boutros
Division Signaling and Functional Genomics and Heidelberg University
Department for Cell and Molecular Biology, Medical Faculty Mannheim
Im Neuenheimer Feld 580
69120 Heidelberg
Germany


Michael Freeley
Department of Clinical Medicine
Trinity College Dublin
Dublin
Ireland


Michael S. Banos
Dharmacon
part of GE Healthcare
2650 Crescent Dr., Suite, 100
Lafayette
CO 80026
USA


Namyoul Kim
Institute Pasteur Korea
696 Sampyeong-dong
Bundang-gu
Gyeonggi-do, 463-400
South Korea


Olivia Perwitasari
Department of Infectious Diseases
University of Georgia, College of Veterinary Medicine
111 Carlton Street
Athens
GA 30602
USA


Paula Brooks
Department of Infectious Diseases
University of Georgia, College of Veterinary Medicine
111 Carlton Street
Athens
GA 30602
USA


Ralph A. Tripp
Department of Infectious Diseases
University of Georgia, College of Veterinary Medicine
111 Carlton Street
Athens
GA 30602
USA


Roderick L. Beijersbergen
Division of Molecular Carcinogenesis, Cancer Genomics Center and Cancer Systems Biology Center
The Netherlands Cancer Institute
1066 CX Amsterdam
The Netherlands


S. Mark Tompkins
Department of Infectious Diseases
University of Georgia
Athens
Georgia
USA


Sabine van der Sanden
Centers for Disease Control and Prevention
600 Clifton Road NE
Atlanta
GA, 30333
USA


Samantha Barichievy
Gene Expression and Biophysics Group
Synthetic Biology Emerging Research Area
Council for Scientific and Industrial Research
Pretoria
South Africa


Sarah B. Anderson
Challenge Technology Inc.
4950 Ward Road
Wheat Ridge
CO 80033
USA


Sean M. Johnston
ICCB-Longwood Screening Facility
Harvard Medical School
250 Longwood Avenue, SGM Building Room 604
Boston
MA 02115
USA


Sinu P. John
Signaling Systems Unit, Laboratory of Systems Biology
National Institute of Allergy and Infectious Diseases, National Institutes of Health
Bethesda
MD 20892
USA


Steven Oberste
Centers for Disease Control and Prevention
1600 Clifton Road NE
Atlanta
GA, 30333
USA


TaeKyu Lee
Institute Pasteur Korea
696 Sampyeong-dong
Bundang-gu
Gyeonggi-do, 463-400
South Korea


Timothy J. Doran
CSIRO Australian Animal Health Laboratory
Geelong 3220
Victoria
Gyeonggi-do
Australia


Veronica Soloveva
Institute Pasteur Korea
696 Sampyeong-dong
Bundang-gu
Gyeonggi-do, 463-400
South Korea


Weilin Wu
Department of Infectious Diseases
University of Georgia, College of Veterinary Medicine
111 Carlton Street
Athens
GA 30602
USA


Yong-Jun Kwon
Institute Pasteur Korea
696 Sampyeong-dong
Bundang-gu
Gyeonggi-do, 463-400
South Korea




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