Guide to Electroporation and Electrofusion

559 Pages·1991·27.095 MB·English

by  Donald C. Chang, Bruce M. Chassy, James A. Saunders and Arthur E. Sowers (Eds.)

Guide to
Electroporation and
Electrofusion
Edlted by
Donald C. Chang
Department of Molecular Physiology and Biophysics
Baylor College of Medicine
Houston, Texas
Department of Biology
Hong Kong University of Science and Technology
Kowloon, Hong Kong
Bruce M. Chassy
Department of Food Science
University of Illinois at Urbana-Champaign
Urbana, Illinois
James A. Saunders
Plant Sciences Institute
Beltsville Agricultural Research Center
United States Department of Agriculture
Beltsville, Maryland
Arthur E. Sowers
Department of Biophysics
School of Medicine
University of Maryland at Baltimore
Baltimore, Maryland
Academic Press, Inc.
Harcourt Brace Jovanovich, Publishers
San Diego New York Boston London Sydney Tokyo Toronto
In Memorium
The editors would like to recognize the untimely passing of Eve Morris, co-founder
and President of BTX, whose death on January 3, 1991 struck us all with a great
sense of professional and personal loss. Pioneering work by BTX, one of several
commercial companies, resulted in the introduction and widespread use of com-
mercial pulse generators for electroporation and electrofusion, which contributed
greatly to the development of this field of research.
This book is printed on acid-free paper. @
Copyright © 1992 by ACADEMIC PRESS, INC.
All Rights Reserved.
No part of this publication may be reproduced or transmitted in any form or by any
means, electronic or mechanical, including photocopy, recording, or any information
storage and retrieval system, without permission in writing from the publisher.
Academic Press, Inc.
San Diego, California 92101
United Kingdom Edition published by
Academic Press Limited
24-28 Oval Road, London NW1 7DX
Library of Congress Cataloging-in-Publication Data
Guide to electroporation and electrofusion / Donald C. Chang ...
[et. al.].
p. cm.
Includes index.
ISBN 0-12-168040-1 (Hardcover)
ISBN 0-12-168041-X (Paperback)
1. Electroporation. 2. Electrofusion. I. Chang Donald C
GH585.5E48H36 1992
574.8-dc20 91-18976
CIP
PRINTED IN THE UNITED STATES OF AMERICA
91 92 93 94 9 8 7 6 5 4 3 2 1
In Memorium
The editors would like to recognize the untimely passing of Eve Morris, co-founder
and President of BTX, whose death on January 3, 1991 struck us all with a great
sense of professional and personal loss. Pioneering work by BTX, one of several
commercial companies, resulted in the introduction and widespread use of com-
mercial pulse generators for electroporation and electrofusion, which contributed
greatly to the development of this field of research.
This book is printed on acid-free paper. @
Copyright © 1992 by ACADEMIC PRESS, INC.
All Rights Reserved.
No part of this publication may be reproduced or transmitted in any form or by any
means, electronic or mechanical, including photocopy, recording, or any information
storage and retrieval system, without permission in writing from the publisher.
Academic Press, Inc.
San Diego, California 92101
United Kingdom Edition published by
Academic Press Limited
24-28 Oval Road, London NW1 7DX
Library of Congress Cataloging-in-Publication Data
Guide to electroporation and electrofusion / Donald C. Chang ...
[et. al.].
p. cm.
Includes index.
ISBN 0-12-168040-1 (Hardcover)
ISBN 0-12-168041-X (Paperback)
1. Electroporation. 2. Electrofusion. I. Chang Donald C
GH585.5E48H36 1992
574.8-dc20 91-18976
CIP
PRINTED IN THE UNITED STATES OF AMERICA
91 92 93 94 9 8 7 6 5 4 3 2 1
Preface
In the last decade, there has been a widespread use of electroporation and electrofusion
in many different fields, including molecular biology, cell biology, plant genetics,
hybridoma technology, agricultural research, and others. The number of published
papers that involved the use of electroporation has grown in an exponential manner
since the mid- 1980s. Consequently, we feel there is a strong need for a comprehensive
treatise on the subjects of electroporation and electrofusion.
This book includes four major parts. Part I deals with the basic principles and
the fundamental processes of electroporation and electrofusion. The chapters in this
part review the recent progress of studies aimed at providing a better understanding
of the molecular mechanisms by which the externally applied electric field is able
to induce membrane permeation and cell fusion. These studies include both theo-
retical and experimental works, and use a wide range of approaches including
physical, chemical, and biological methods.
In Part II, important applications of electroporation and electrofusion in many
different areas of biological research are reviewed. These applications include gene
transfer in mammalian cells, genetic manipulation in plant cells, genetic transfor-
mation of bacteria and yeast, electroinjection of exogenous molecules for studying
cellular functions, and production of hybridoma and human monoclonal antibodies.
Other innovative applications such as embryo cloning, electroinsertion of proteins
into cell membranes, and electrofusion of cells to tissues are also included.
The chapters of Part III are a collection of practical protocols. This section provides
useful information to assist readers in designing experiments using electroporation
and electrofusion. Step-by-step procedures are provided for the use of electroporation
and electrofusion to introduce genes (and other molecules) into various biological
cells, including mammalian cells, plant cells, bacteria, and yeasts. Methods to
improve hybridoma production by electrofusion are also provided in some of these
chapters.
Finally, in Part IV, the instrumentation required for electroporation and elec-
trofusion is discussed. We have summarized the features of most of the commercially
available equipment designed for electroporation and electrofusion. Some useful tips
for optimizing the operation of this equipment are also provided.
This book is written to satisfy the needs of scientists in many different fields and
at different levels of experience. For the beginners, this book can serve as a practical
laboratory manual; for the research scientist, it can serve as a guide for experimental
design, particularly in the development of research proposals; and for experienced
IX
χ Preface
scientists in this field, the book will serve as a current overview of the ongoing
research in leading laboratories around the world.
Chapters in the book are written by top experts in the studies of electroporation
and electrofusion. The authors include not only most of the well-known pioneers
in these fields, but also many international authorities whose work has provided the
most recent understanding of the basic mechanisms of electroporation and electro-
fusion. Chapters in Part II and Part III are contributed by leading scientists who
are largely responsible for the development of many of the applications of electro-
poration and electrofusion in various areas of biological research. Because of their
collective efforts, this book is able to provide an extraordinary breadth and depth of
coverage.
One important reason we were able to attract so many top experts to contribute
to this book was that the editors were involved in organizing the first International
Conference on Electroporation and Electrofusion held at Woods Hole, Massachusetts,
in late 1990. Without the impetus provided by the conference, it would have been
nearly impossible to recruit all the experts to contribute to this book. We would
like to gratefully acknowledge Life Technologies, Bio-Rad Laboratories, BTX, Inc.,
Hoefer Scientific Instruments, IBI/Kodak, the U. S. Department of Agriculture,
and the Marine Biological Laboratory of Woods Hole. We also want to thank
Huntington Potter and T. Y. Tsong, who made valuable contributions. Finally,
the editors appreciate the support by Lorraine Lica of Academic Press.
Overview of Electroporation
and Electrofusion
Donald C. Chang, 1 James A. Saunders, 2 Bruce M. Chassy, 3
and Arthur E. Sowers 4
department of Molecular Physiology and Biophysics, Baylor College of Medicine,
Houston, Texas 77030
2Plant Sciences Institute, Beltsville Agricultural Research Center, United States
Department of Agriculture, Beltsville, Maryland 20705
3Department of Food Science, University of Illinois, Urbana, Illinois 61801
4Department of Biophysics, School of Medicine, University of Maryland at Baltimore,
Baltimore, Maryland 21201
I. Introduction
A. Electroporation
B. Electrofusion
II. Advantages of Electroporation and Electrofusion
III. Mechanisms of Electroporation and Electrofusion
IV. Applications of Electroporation
V. Application of Electrofusion
References
I. Introduction
A. Electroporation
Electroporation is a phenomenon in which the membrane of a cell exposed to high-
intensity electric field pulses can be temporarily destabilized in specific regions of
the cell. During the destabilization period, the cell membrane is highly permeable
to exogenous molecules present in the surrounding media.
!Present address: Department of Biology, Hong Kong University of Science and Technology, Kow-
loon, Hong Kong.
Guide to Electroporation and Electrofusion
Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved. 1
2 Donald C. Chang et al.
Electroporation thus can be regarded as a massive microinjection technique that
can be used to inject a single cell or millions of cells with specific components in
the culture medium.
Several publications appeared in the 1950s and 1960s that showed that an ex-
ternally applied electric field can induce a large membrane potential at the two poles
of the cell (Cole, 1968). It was known that an excessively high field could also cause
cell lysis (Sale and Hamilton, 1967; Sale and Hamilton, 1968). By the early 1970s,
several laboratories had found that when the induced membrane potential reaches
a critical value, it can cause a dielectric breakdown of the membrane. Such breakdown
was demonstrated in red blood cells (Crowley, 1973; Zimmermann et al., 1974)
and in model membranes (Neumann and Rosenheck, 1972; Neumann and Rosen-
heck, 1973).
By the late 1970s, the concept of "membrane pore" formation or membrane
destabilization, as a result of dielectric breakdown of the cell membrane, was formally
discussed (Kinosita and Tsong, 1977). At about the same time, it was found that
if the electric field was applied as a very short duration pulse, the cells could recover
from the electrical treatment. This implied that these electric field-mediated "pores"
were resealable and could be induced without permanent damage to the cells (Baker
and Knight, 1978a; 1978b; Gauger and Bentrup, 1979; Zimmermann et al., 1980).
By the early 1980s, reports began appearing that showed that many small mol-
ecules, such as sucrose, dyes, or monovalent or divalent ions, could pass through
these electric field-induced "membrane pores" to a broad array of cell types. Many
laboratories started to use pulsed electric fields to introduce a variety of molecules
into the cells, including drugs (Zimmermann et al., 1980), catacholamine and Ca-
EGTA (Knight and Baker, 1982), and DNA (Wong and Neumann, 1982; Neumann
et al., 1982; Potter et al., 1984; Fromm et al., 1986). In the last decade, there has
been an explosion in the number of groups using this "electroporation" technique
to incorporate various molecules into many different types of cells. Recently, a new
method of electroporation which utilizes a pulsed radio-frequency electric field to
break down the cell membrane has been developed (Chang, 1989).
B. Electrofusion
When neighboring cells are brought into contact during the electrically mediated
membrane destabilization process outlined above, these cells can be induced to fuse.
The number of cells that can be fused by the application of a pulse (or pulses) of
this high-intensity electric field is dependent on the size and type of cell, as well
as the field intensity of the electrical pulse. The experimental procedures are very
similar to those of electroporation, except that the cells to be fused must be brought
into contact first. This cell contact can be accomplished by (1) mechanical manip-
ulation, (2) chemical treatment, or (3) dielectrophoresis (in which the cells are lined
up in chains by applying a low-intensity, high-frequency, oscillating electric field).
Chapter 1 Overview of Electroporation and Electrofusion 3
The phenomenon of electrofusion is closely related to that of electroporation.
Late in 1979 and early in the 1980s several laboratories had reported success in
using electrical pulses to induce fusion in various systems, including plant cells
(Senda et al., 1979; Zimmermann and Scheurich, 1981) and red blood cells (Scheu-
rich et al., 1980). One significant contribution of Zimmermann's group was their
utilization of the phenomenon of dielectrophoresis (Pohl, 1978) to facilitate cell
contact, thus making the electrofusion method more widely useful. Since the be-
ginning of the 1980s, "electrofusion" has been applied to fuse many different cell
types, and has become the method of choice for cell hybridization.
II. Advantages of Electroporation and Electrofusion
The pulsed electric field method has a number of advantages over the conventional
methods of cell permeabilization or cell fusion. It is a noninvasive, nonchemical
method that does not seem to alter the biological structure or function of the target
cells. Electrofusion is relatively easy to perform and is much more time efficient
than the traditional chemical or biological fusion techniques. Also, unlike the other
chemical or biological methods, the electric field method can be relatively nontoxic.
The efficiency of the electric field method is generally significantly better than most
alternative methods, and finally, because the electric field method is a physical
method, it can be applied to a much wider selection of cell types.
III. Mechanisms of Electroporation and Electrofusion
The basic phenomenology of electroporation and electrofusion are reasonably well
known, although the molecular mechanisms by which the electric field interacts
with the cell membrane are still under active investigation. Basically, a membrane
potential is induced by the externally applied electric field. The electrical field is
usually induced by a relatively short DC pulse. The pulse can be either a square-
wave pulse, usually with a duration of less than 100 μ$, or it can be an exponentially
decaying capacitive discharge pulse with a duration in the millisecond range (Saun-
ders et al., 1989).
When the induced potential reaches a critical value, it causes an electrical break-
down of the cell membrane. The value of this critical potential is about 1 V, but
can vary depending on the pulse width, composition of membrane, etc. Multiple
membrane pores are formed as a result of breakdown. Many studies have been done
to characterize the structure and properties of these electropores (see Part I of this
book). Very recently, porelike structures have been visualized for the first time in
red blood cells using a rapid-freezing microscopy technique (see Chapter 2 of this
book). The dynamics of pore formation and resealing are also under active inves-
tigation at this time (See Part I). The mechanisms by which membranes of neigh-
4 Donald C. Chang et al.
boring cells are induced to fuse by the electric field is not yet clearly understood,
but several theories have been proposed (See Chapters 6, 7, 8, 10, and 11).
Issues to be resolved include: Does the applied field cause a reversible or irre-
versible breakdown of the cell membrane? Does electroporation or electrofusion
occur exclusively at the lipid bilayer region of the cell membrane? In other words,
what is the role of membrane proteins?
IV. Applications of Electroporation
The applications of electroporation or electrically mediated gene transfer techniques
are responsible for the major part of the popularity of this rapidly expanding field.
The ability of a high-voltage pulse to reversibly change the permeability of the cell
membrane leaves the tantalizing possibility of incorporating specific genes into
relatively large numbers of isolated cells. Although it is not 100% effective, trans-
formation yields as high as 60—70% have been obtained with some regularity
(Saunders, et al., 1989). Different researchers have used a variety of names to describe
the electrically mediated gene transfer processes, including electroinjection, elec-
trotransfection, and electrical microinjection, as well as electroporation, but the
basic process is similar in all cases.
Specific applications for electroporation have involved the introduction of both
DNA and RNA to a variety of plant, animal, bacterial, and yeast cells. Although
marker genes were originally the most popular type of DNA to be incorporated into
the recipient cells, recent trends have used functional genes that are important to
biotechnology. Other major applications are injection of drugs, proteins, metabo-
lites, molecular probes, and antibodies for studies of cellular structure and function.
V. Applications of Electrofusion
The applications of electrofusion extend into many different areas using a wide
variety of cell types. In plants, where individual cells have the potential to regenerate
into mature differentiated tissue, somatic hybridization of isolated protoplasts by
electrofusion has been a popular method of genomic gene transfer. This is an ex-
tremely efficient method of cell fusion, which results in relatively high yields of
multinucleate cells containing the entire combined genomes of each parental cell
type. Unfortunately, fusions among each of the parental cell types are as common,
if not more so, than fusions between the different parental cell types. Thus, the
selection of the hybrid cell of choice is an integral part of any plant fusion protocol.
A second area that has gained considerable interest in electrofusion research has
been that of hybridoma/monoclonal antibody production. The selection system for
the proper fusion partners, that is, antibody production, is already built into the
Chapter 1 Overview of Electroporation and Electrofusion 5
system. Harvested cells producing the desired antibody can be collected in culture,
processed, and relatively large amounts of antibodies recovered. The use of electro-
fusion techniques in this application has sometimes improved the yields and re-
coverability of hybridoma cells by 100-fold in comparison to chemical fusion meth-
ods.
Another exciting area of electrofusion that is just emerging is the area of cell/tissue
electrofusion. Experimental protocols in which isolated cells are electrofused to
various tissue either in vitro or in some cases in vivo, are being used to effect genetic
transformations that were previously not possible (Heller and Gilbert, Chapter 24
of this book).
In summary, the electroporation and electrofusion techniques are highly versatile
and widely useful physical methods that have tremendous potential applications in
cell biology, molecular biology, biotechnology, and other branches of biological
research.
References
Baker, P. F., and Knight, D. E. (1978a). A high voltage technique for gaining rapid access
to the interior of secretory cells. J. Physiol. 284, 30.
Baker, P. F., and Knight, D. E. (1978b). Influence of anions on exocytosis in leaky bovine
adrenal medullary cells. J. Physiol. 296, 106.
Chang, D. C. (1989). Cell poration and cell fusion using an oscillating electric field. Biophys.
J. 56, 641-652.
Cole, K. S. (1968). A chapter of classical biophysics. In "Membranes, Ions, and Impulses."
University of California Press, Berkeley, pp. 12-18.
Crowley, J. M. (1973). Electrical breakdown of bimolecular lipid membranes as an electro-
mechanical instability. Biophys. J. 13, 711-724.
Fromm, M. L., Taylor, P., and Walbot, V. (1986). Stable transformation of maize after
gene transfer by electroporation. Nature 319, 791-793.
Gauger, B., and Bentrup, F. W. (1979). A study of dielectric membrane breakdown in the
Fucus egg. J. Membrane Biol. 48, 249-264.
Kinosita, Κ., Jr., and Tsong, T. Y. (1977). Hemolysis of human erythrocytes by a transient
electric field. Proc. Natl. Acad. Sei. USA 74, 1923-1927.
Knight, D. E., and Baker, P. F. (1982). Calcium dependence of catecholamine release from
bovine adrenal medullary cells after exposure to intense electric fields. J. Membrane Biol.
68, 107-140.
Neumann, E., and Rosenheck, Κ. (1972). Permeability changes induced by electric impulses
in vesicular membranes. J. Membrane Biol. 10, 279-290.
Neumann, E., and Rosenheck, Κ. (1973). Potential difference across vesicular membranes.
J. Membrane Biol. 14, 194-196.
Neumann, E., Schaefer-Ridder, M., Wang, Y., and Hofschneider, P. H. (1982). Gene
transfer into mouse myloma cells by electroporation in high electric fields. ΕΜΒ0 J. 1,
841-845.
Pohl, Η. Α. (1978). "Dielectrophoresis." Cambridge University Press, London.