Vision Research Protocols M E T H O D S I N M O L E C U L A R M E D I C I N ETM John M. Walker, SERIES EDITOR 60. Interleukin Protocols, edited by Luke 47. Vision ResearchProtocols,edited by A. J. O’Neill and Andrew Bowie, 2001 P. Elizabeth Rakoczy, 2001 59. Molecular Pathology of the Prions, 46. Angiogenesis: Reviews and edited by Harry F. Baker, 2001 Protocols,edited byJ. Clifford Murray, 2001 58. Metastasis Research Protocols: Volume 2, Cell Behavior In Vitro 45. Hepatocellular Carcinoma: Methods and In Vivo, edited by Susan A. and Protocols, edited by Nagy A. Brooks and Udo Schumacher, 2001 Habib, 2000 57. Metastasis Research Protocols: 44. Asthma: Mechanisms and Protocols, Volume 1, Analysis of Cells and edited by K. Fan Chung and Ian Tissues,edited by Susan A. Brooks Adcock, 2000 andUdo Schumacher, 2001 43. Muscular Dystrophy: Methods and 56. Human Airway Inflammation: Protocols,edited by Katherine B. Sampling Techniques and Analytical Bushby and Louise Anderson, 2000 Protocols,edited by Duncan F. Rogers 42. Vaccine Adjuvants: Preparation and Louise E. Donnelly, 2001 Methods and Research Protocols, edited by Derek T. O’Hagan, 2000 55. Hematologic Malignancies: Methods and Protocols, edited by Guy B. Faguet, 41. Celiac Disease: Methods and 2001 Protocols, edited by Michael N. Marsh,2000 54. Mycobacterium Tuberculosis Protocols,edited by Tanya Parish 40. Diagnostic and Therapeutic and Neil G. Stoker, 2001 Antibodies,edited by Andrew J. T. George and Catherine E. Urch, 2000 53. Renal Cancer: Methods and Protocols, edited by Jack H. Mydlo, 2001 39. Ovarian Cancer: Methods and Protocols, edited by John M. S. Bartlett, 2001 52. Atherosclerosis Methods and Protocols, edited by Angela F. Drew, 2001 38. Aging Methods and Protocols, edited by Yvonne A. Barnett and 51. Angiotensin Protocols, edited by Christopher R. Barnett, 2001 Donna H. Wang, 2001 37. Electrochemotherapy, 50. Colorectal Cancer: Methods and Electrogenetherapy, and Transdermal Protocols, edited by Steven M. Drug Delivery: Electrically Mediated Powell,2001 Delivery of Molecules to Cells, edited by 49. Molecular Pathology Protocols, Mark J. Jaroszeski, Richard Heller, and edited by Anthony A. Killeen, 2001 Richard Gilbert, 2000 48. Antibiotic Resistance Methods and 36. Septic Shock Methods and Protocols, Protocols,edited by Stephen H. edited by Thomas J. Evans, 2000 Gillespie,2001 M E T H O D S I N M O L E C U L A R M E D I C I N ETM Vision Research Protocols Edited by P. Elizabeth Rakoczy Centre for Ophthalmology and Visual Science, Lions Eye Institute, University of Western Australia, Nedlands, Perth Humana Press Totowa, New Jersey © 2001 Humana Press Inc. 999 Riverview Drive, Suite 208 Totowa, New Jersey 07512 All rights reserved. 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Printed in the United States of America. 10 9 8 7 6 5 4 3 2 1 Library of Congress Cataloging in Publication Data Vision research protocols / edited by P. Elizabeth Rakoczy p.; cm.--(methods in molecular medicine; 47) Includes bibliographical references and index. ISBN 0-89603-837-8 (alk. paper) 1. Ophthalmology--Research--Methodology. 2. Molecular biology--Methodology. 3. Eye--Diseases--Molecular aspects. 4. Eye--Diseases--Gene therapy. I. Rakoczy, P. Elizabeth. II. Series. [DNLM: 1. Eye Diseases--genetics. 2. Eye Diseases--physiopathology. 3. Genetics, Biochemical. WW 140 O207 2000] RE58 .O25 200 00-033502 617.7--dc21 CIP Preface Genetic technology is the buzzword of the new millennium. Via the media, we are informed day by day about new genes, disease-causing mutations, cloned animals, “Frankenstein” food, and further advances in the Human Genome Project. However, in spite of the constant media attention, most people remain con- fused about the importance and application of these new discoveries. It seems that only the high priests of this new technology, armed with PhDs in molecu- lar biology, understand the meaning, importance, and consequences of these new breakthroughs. But should it be like this? After all, molecular biology is only a tool, though one that enables us to study the secrets of life, death, and disease development. Vision Research Protocolshas been written for thosescientists, optometrists, and ophthalmologists who are interested in eye research, but have not been trained in molecular biology. It covers molecular biological techniques from the basics to the most sophisticated recent technologies. In each case, the tech- niques described have been adapted to the special requirements of eye re- search. The first four chapters discuss crucial molecular biological methodologies that create a basis for the more complex methodologies pre- sented later. The last chapter discusses the impact of these new technologies on everyday clinical services. The first four chapters follow the basic dogma of molecular biology. They describe the extraction of DNA and detection of mRNA and protein expression in vitro and in vivo. In addition, Chapter 2 gives a description of the basics of tissue culture technology, which has significantly advanced bio- logical research since the 1970s. In a philosophical sense, diseases can be considered as disturbances in the normal balance within a complex organism. In molecular terms, certain genes become up or down regulated, or turned on and off out of sequence. In this respect, the new molecular biological techniques can have a significant impact both in the development of animal models and on new treatments for diseases. Chapters 5, 6, and 12 discuss technologies suitable for the down regulation of gene expression. Chapters 7–10 are devoted to the new gene therapy technologies. Following a very enthusiastic start in the early 1990s, v vi Preface gene therapy proved to be a difficult technology to apply, and further improve- ments are required before it can be considered ready for clinical application. However, even in its present form, it is particularly suitable for the develop- ment of animal models and for testing new therapies in transgenic and knock- out animal models. Vision Research Protocolscontains a detailed description of the three most popular viral delivery vehicles: recombinant adeno, adeno- associated, and retroviruses. In addition, delivery methods for the most popu- lar animal models are also described. In summary, I recommend Vision Research Protocolsto all medical and nonmedical scientists who are thinking of using these new technologies in their research, or those clinicians who would like to find out the current state of progress in this important field of molecular medicine. I would like to sincerely thank all the contributors, who are leading sci- entists in their fields with real hands-on experience in the different techniques. They were wonderful to work with, and I would specifically thank them for providing their invaluable comments in the troubleshooting sections of each chapter. I also would like to thank Ms. Louise Kemp, who has been superb in organizing the contributions of experts from three continents. P. Elizabeth Rakoczy Contents Preface .............................................................................................................v Contributors.....................................................................................................ix 1 Basic Molecular Biology Techniques Chooi-May Lai........................................................................................1 2 Transient Transfection of Human Retinoblastoma Cells: Application to the Analysis of the Regulatory Regions of Photoreceptor-Specific Genes Debora B. Farber, Leonid E. Lerner, and Andrea S. Viczian..........31 3 In Situ Hybridization Xian-Jie Yang.......................................................................................45 4 Immunocytochemical Studies of the Retina Ann H. Milam........................................................................................71 5 Antisense DNA Technology Piroska Elizabeth Rakoczy.................................................................89 6 Inhibition of Gene Expression by Ribozymes William W. Hauswirth, Lynn C. Shaw, Patrick O. Whalen, Jason J. Fritz, D. Alan White, and Alfred S. Lewin...................105 7 Intraocular Delivery of Recombinant Virus Fong-Qi Liang, Vibha Anand, Albert M. Maguire, and Jean Bennett...........................................................................125 8 Preparation of Recombinant Adenoviruses Karl G. Csaky......................................................................................141 9 Generation of Recombinant Adeno-Associated Virus Fabienne Rolling................................................................................157 10 Preparation of Recombinant Retroviruses Xian-Jie Yang.....................................................................................171 11 Generation of Transgenic Mice for Studies of Ocular Development and Disease Abha R. Gupta, Nadine S. Dejneka, Albert M. Maguire, and Jean Bennett...........................................................................191 vii viii Contents 12 Generation of Knockout Animal Models T. Michael Redmond..........................................................................215 13 Mutation Analysis of Ocular Genes Michael Danciger...............................................................................237 14 Ophthalmologists' Perspective Susan M. Downes, N. H. Victor Chong, and Alan C. Bird.............251 Index............................................................................................................307 Contributors VIBHA ANAND • Chance Labs, Department of Ophthalmology, University of Pennsylvania, Philadelphia, PA JEAN BENNETT • Scheie Eye Institute, Department of Ophthalmology, University of Pennsylvania, Philadelphia, PA ALAN C. BIRD • Medical Retina Department, Moorfields Eye Hospital, London, UK N. H. VICTOR CHONG • Medical Retina Department, Moorfields Eye Hospital, London, UK KARL G. CSAKY • Laboratory of Immunology, NEI/NIH, Bethesda, MD MICHAEL DANCIGER • Department of Biology, Loyola Marymount University, and Jules Stein Eye Institute, UCLA School of Medicine, Los Angeles, CA NADINE S. DEJNEKA • Department of Ophthalmology, University of Pennsylvania, Philadelphia, PA SUSAN M. DOWNES • Medical Retina Department, Moorfields Eye Hospital, London, UK DEBORA B. FARBER • Jules Stein Eye Institute, UCLA School of Medicine, Los Angeles, CA JASON J. FRITZ • Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL ABHA R. GUPTA • Department of Ophthalmology, University of Pennsylvania, Philadelphia, PA WILLIAM W. HAUSWIRTH • Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL CHOOI-MAY LAI • Lions Eye Institute, University of Western Australia, Nedlands, Perth, Western Australia LEONID E. LERNER •Jules Stein Eye Institute, UCLA School of Medicine, Los Angeles, CA ALFRED S. LEWIN •Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL FONG-QI LIANG • Chance Labs, Department of Ophthalmology, University of Pennsylvania, Philadelphia, PA ALBERT M. MAGUIRE • Department of Ophthalmology, University of Pennsylva- nia, Philadelphia, PA ix