Table Of ContentM 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
VViissiioonn
RReesseeaarrcchh
PPrroottooccoollss
EEddiitteedd bbyy
PP.. EElliizzaabbeetthh RRaakkoocczzyy
HHuummaannaa PPrreessss
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. No part of this book may be reproduced, stored in a retrieval system, or transmitted in
any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise
without written permission from the Publisher. Methods in Molecular Medicine™is a trademark of The
Humana Press Inc.
All authored papers, comments, opinions, conclusions, or recommendations are those of the author(s), and
do not necessarily reflect the views of the publisher.
Cover design by Patricia F. Cleary
This publication is printed on acid-free paper. (cid:39)
ANSI Z39.48-1984 (American Standards Institute) Permanence of Paper for Printed Library Materials.
For additional copies, pricing for bulk purchases, and/or information about other Humana titles, contact
Humana at the above address or at any of the following numbers: Tel.: 973-256-1699; Fax: 973-256-8341;
E-mail: [email protected] or visit our Website: http://humanapress.com
Photocopy Authorization Policy:
Authorization to photocopy items for internal or personal use, or the internal or personal use of specific
clients, is granted by Humana Press Inc., provided that the base fee of US $10.00 per copy, plus US $00.25
per page, is paid directly to the Copyright Clearance Center at 222 Rosewood Drive, Danvers, MA 01923.
For those organizations that have been granted a photocopy license from the CCC, a separate system of
payment has been arranged and is acceptable to Humana Press Inc. The fee code for users of the Transactional
Reporting Service is: [0-89603-837-8/00 $10.00 + $00.25].
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
