Gene Silencing by RNA Interference: Technology and Application PDF
Preview Gene Silencing by RNA Interference: Technology and Application
Gene Silencing by RNA Interference Technology and Application Copyright 2005 by CRC Press LLC Gene Silencing by RNA Interference Technology and Application EDITED BY Muhammad Sohail CRC PR ESS Boca Raton London New York Washington, D.C. Copyright 2005 by CRC Press LLC Gene Silencing by RNA Interference Copyright 2005 by CRC Press LLC Sohail-2141-FM.pmd 1 7/26/2004, 9:11 AM Gene Silencing by RNA Interference: Technology and Application edited by Muhammad Sohail Copyright 2005 by CRC Press LLC Sohail-2141-FM.pmd 3 7/26/2004, 9:11 AM Library of Congress Cataloging-in-Publication Data Gene silencing by RNA interference : technology and application / edited by Muhammad Sohail. p. cm. Includes bibliographical references and index. ISBN 0-8493-2141-7 (alk. paper) 1. Gene silencing—Laboratory manuals. 2. RNA—Laboratory manuals. I. Sohail, Muhammad. QH450. G4635 2004 660.6'5—dc22 2004051934 This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. A wide variety of references are listed. Reasonable efforts have been made to publish reliable data and information, but the author and the publisher cannot assume responsibility for the validity of all materials or for the consequences of their use. Neither this book nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording, or by any information storage or retrieval system, without prior permission in writing from the publisher. All rights reserved. Authorization to photocopy items for internal or personal use, or the personal or internal use of specific clients, may be granted by CRC Press LLC, provided that $1.50 per page photocopied is paid directly to Copyright Clearance Center, 222 Rosewood Drive, Danvers, MA 01923 USA. 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Visit the CRC Press Web site at www.crcpress.com © 2005 by CRC Press LLC No claim to original U.S. Government works International Standard Book Number 0-8493-2141-7 Library of Congress Card Number 2004051934 Printed in the United States of America 1 2 3 4 5 6 7 8 9 0 Printed on acid-free paper Copyright 2005 by CRC Press LLC Sohail-2141-FM.pmd 4 7/27/2004, 4:15 PM Foreword Every so often, a new research tool emerges which reshapes an entire field of study. It has become especially clear that recent progress in the biological sciences was driven by technical innovation; Sanger’s sequencing method laid the foundation for genomics and all that has followed. RNAi is one of the most recent entries. Although described several years ago, it has only recently been widely adapted. But it has set in motion intense activity di- rected at discovering the function of genes and, hence, understanding the complex mechanisms of biological systems. It is a remarkable process. Who could have imag- ined that nature would invent a system for the destruction of single-stranded RNAs that was triggered by their double-stranded counterparts? Evidence for the system was hard won. And much work went into unravelling its complexities, which are still not fully understood. However, its power and its usefulness are now proven. Applications will grow. It will in all likelihood become a routine technology in many areas of biology. But much needs to be done to achieve that status. It is a method that is applied to organisms, each of which will have its own quirks and foibles; i.e., the nematode worm consumes RNAi agents in its normal diet of bacteria, a system of delivery that is unlikely to work in many animals. To advance the technology, we need a deeper understanding of the molecular mechanisms that produce the effect; however, these may differ in different organisms, even in different cells of the same organism. Above all, we need estab- lished methods that work in the hands of non-specialists who want to apply them to their experiments, and in the world of high-throughput biology, we would like to have methods that can be automated to apply on the large scale. We are fortunate to live in an age which accords to technology development the respect it deserves. Funding agencies give their support to technology development and inventors get their rewards for innovation; there are even signs that elite scientific journals are catching up with the trend. The chapters of this volume, written by scientists at the leading edge, illustrate the present state of the art in the RNAi field. They provide the basic knowledge needed by those who want to apply the method to their own system, to those who want to understand the biology of the RNAi mechanism, and to those who want to engage in furthering this imoportant technology. SIR EDWIN M. SOUTHERN, FRS Oxford Copyright 2005 by CRC Press LLC Sohail-2141-FM.pmd 5 7/27/2004, 4:15 PM Preface Gene silencing induced by RNA interference is one of the most topical and exciting subjects in molecular biology today. It offers new ways of quickly and easily creating loss-of-function phenotypes. Therefore, molecular biologists in almost every disci- pline are keen to employ this tool in their model systems/organisms. DNA sequencing projects are revealing a wealth of information that needs to be analyzed and under- stood before it can be implemented. To do so, the functions of several thousands of genes need to be elucidated and that requires high-throughput techniques. Conven- tional gene knockout techniques are slow and cumbersome and are unlikely to meet the high demands of such analyses. Antisense technologies hold the potential of pro- viding a high throughput alternative to gene knockout methods. However, there have been considerable difficulties in effectively applying conventional antisense reagents for such purpose, such as the lack of target specificity and low efficacy. The advent of RNA interference tools is likely to solve at least some of these difficulties. RNA interference appears to be highly conserved and occurs in both plants and animals. Artificial silencing of an endogenous gene can be triggered by the introduc- tion of short or long double-stranded RNA, a trans-gene, or a virus. Methodologies for using RNA interference in plant, insect, and mammalian cells have been devel- oped and have been published in journal articles over the past few years. However, due to space constraints, it is not always possible for the authors to provide full details of methods, and the often sketchy methodological information can be difficult to fol- low. Thus, a reliable, well-researched resource that combines the essential background to the subject, with tried and tested practical techniques and troubleshooting advice was needed. This book is intended to fill the gaps, and to be useful to both beginners and experienced users in the fields, in academia, and in the commercial sector. This volume covers a wide variety of topics — from design and production of siRNAs to their application in cultured cells, embryos, animals, and plants. Experts in the field who either invented the methods or have extensive experience in using them have written the chapters. Each chapter first provides necessary background to the subject area and then gives detailed methods in an easy-to-follow step-by-step fash- ion, with troubleshooting hints and useful tips. The first chapter provides a general introduction to the subject area. Chapters 2 through 8 focus on the various methods of siRNA design, chemical and enzymatic production, and plasmid- and virus-mediated intracellular expression. Chapter 9 provides valuable information on improving and monitoring exogenous delivery of siRNAs into cells. The remaining chapters deal with the application of RNAi tools in a number of biological systems including cul- tured cells, human parasites, embryos, model animals, and plants. MUHAMMAD SOHAIL Oxford Copyright 2005 by CRC Press LLC Sohail-2141-FM.pmd 7 7/27/2004, 4:15 PM Acknowledgments I am highly indebted to all the authors for their hard work and support in bringing this challenging project to completion and consider it an honor to have such a prestigious and world-class team. I am also grateful to my colleagues at CRC Press, Barbara Norwitz, Judith Spiegel, Erika Dery, Barbara Uetrecht-Pierre, and their teams, for their considerable patience, timely assistance and advice, and highly efficient pro- cessing of the book — from the proposal submission stages to print. Last but not least, I am most thankful to my parents for their continuous support and prayers and my children and my wife for their immense patience and support throughout the course of this project. Copyright 2005 by CRC Press LLC Sohail-2141-FM.pmd 9 7/27/2004, 4:15 PM Editor Muhammad Sohail is a senior researcher at the Department of Biochemistry, Uni- versity of Oxford, which he joined in 1995. His major research interest is the study of nucleic acid interactions using oligonucleotide arrays, with particular focus on the design strategies of gene silencing reagents. He obtained a BSc degree in Biochemical Sciences from the University of Punjab in 1986. He then joined Quaid-i-Azam Uni- versity and earned an MSc in Biological Sciences in 1989 and an MPhil in Molecular Biology in 1991. Later in 1991, Dr. Sohail moved to the University of Oxford to study for a doctoral degree and joined the Department of Biochemistry, graduating in 1994. Copyright 2005 by CRC Press LLC Sohail-2141-FM.pmd 11 7/27/2004, 4:15 PM Contributors Geoffrey W. Abbott Frank Buchholz Weill Medical College of Cornell Max Planck Institute of Molecular Cell University Biology and Genetics New York, New York Dresden, Germany Saghir Akhtar Natasha J. Caplen Cardiff University National Cancer Institute Cardiff, United Kingdom Bethesda, Maryland Dong Sung An Virander S. Chauhan University of California–Los Angeles International Center for Genetic Los Angeles, California Engineering and Biotechnology New Delhi, India Peter J. Appleford Irvin S.Y. Chen University of Oxford University of California–Los Angeles Oxford, United Kingdom Los Angeles, California Vincent C. Auyeung Palakodeti V.N. Dasaradhi California Institute of Technology International Center for Genetic Pasadena, California Engineering and Biotechnology New Delhi, India David Baltimore California Institute of Technology Olivier Donzé Pasadena, California Apotech Corporation Université de Genève M. Barciszewska-Pacak Genève, Switzerland Adam Mickiewicz University Poznań, Poland Graeme Doran University of Oxford Mónica Bettencourt-Dias Oxford, United Kingdom University of Cambridge Cambridge, United Kingdom David Drechsel Max Planck Institute of Molecular Cell Queta Boese Biology and Genetics Dharmacon, Inc. Dresden, Germany Lafayette, Colorado Mark E. Drew Anke Brüning-Richardson Washington University School of University of Leeds Medicine Leeds, United Kingdom St. Louis, Missouri Copyright 2005 by CRC Press LLC Sohail-2141-FM.pmd 13 7/27/2004, 4:15 PM
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