Guide to Electroporation and Electrofusion is designed to serve the needs of students, experienced researchers, and newcomers to the field. It is a comprehensive manual that presents, in one source, up-to-date, easy-to-follow protocols necessary for efficient electroporation and electrofusion of bacteria, yeast, and plant and animal cells, as well as background information to help users optimize their results through comprehension of the principles behind these techniques.
Key Features
* Covers fundamentals of electroporation and electrofusion in detail
* Molecular events
* Mechanisms
* Kinetics
* Gives extensive practical information
* The latest applications
* Controlling parameters to maximize efficiency
* Available instrumentation
* Presents applications of electroporation and electrofusion in current research situations
* State-of-the-art modifications to electrical pulses and generators
* Application of electroporation and electrofusion to unique, alternative cell and tissue types
* Gives straightforward, detailed, easy-to-follow protocols for
* Formation of human hybridomas
* Introduction of genetic material into plant cells and pollen
* Transfection of mammalian cells
* Transformation of bacteria, plants, and yeast
* Production of altered embryos
* Optimization of electroporation by using reporter genes
* Comprehensive and up-to-date
* Convenient bench-top format
* Approximately 125 illustrations complement the text
* Complete references with article titles
* Written by leading authorities in electroporation and electrofusion
Electroporation is an efficient method to introduce macromolecules such as DNA into a wide variety of cells. Electrofusion results in the fusion of cells and can be used to produce genetic hybrids or hybridoma cells.Guide to Electroporation and Electrofusion is designed to serve the needs of students, experienced researchers, and newcomers to the field. It is a comprehensive manual that presents, in one source, up-to-date, easy-to-follow protocols necessary for efficient electroporation and electrofusion of bacteria, yeast, and plant and animal cells, as well as background information to help users optimize their results through comprehension of the principles behind these techniques. Covers fundamentals of electroporation and electrofusion in detail: Molecular events, Mechanisms, Kinetics, Gives extensive practical information, The latest applications, Controlling parameters to maximize efficiency, Available instrumentation Presents applications of electroporation and electrofusion in current research situations State-of-the-art modifications to electrical pulses and generators Application of electroporation and electrofusion to unique, alternative cell and tissue types Gives straightforward, detailed, easy-to-follow protocols for Formation of human hybridomas Introduction of genetic material into plant cells and pollen Transfection of mammalian cells Transformation of bacteria, plants, and yeast Production of altered embryos Optimization of electroporation by using reporter genes Comprehensive and up-to-date Convenient bench-top format Approximately 125 illustrations complement the text Complete references with article titles Written by leading authorities in electroporation and electrofusion
Front Cover 1
Guide to Electroporation and Electrofusion 4
Copyright Page 5
Table of Contents 6
Preface 10
Chapter 1. Overview of Electroporation and Electrofusion 12
I. Introduction 12
II. Advantages of Electroporation and Electrofusion 14
III. Mechanisms of Electroporation and Electrofusion 14
IV. Applications of Electroporation 15
V. Applications of Electrofusion 15
Part I: Mechanisms and Fundamental Processes in Electroporation and Electrofusion 18
Chapter 2. Structure and Dynamics of Electric Field-Induced Membrane Pores as Revealed by Rapid-Freezing Electron Microscopy 20
I. Introduction 21
II. Methods 23
III. Results 25
IV. Discussion 33
Chapter 3. Events of Membrane Electroporation Visualized on a Time Scale from Microsecond to Seconds 40
I. Introduction 41
II. Visualization of Transmembrane Potential 41
III. Electroporation Revealed in the Behavior of Transmembrane Potential 45
IV. Large-Hole Formation and Deformation in Giant Liposomes 49
V. Permeation across Porated Membranes 52
VI. Summary 55
Chapter 4. Time Sequence of Molecular Events in Electroporation 58
I. Introduction 58
II. Primary Events of Electroporation 59
III. Other Effects of Pulsed Electric Fields 65
IV. Conclusion 69
Acknowledgments 69
References 69
Chapter 5. Electropores in Lipid Bilayers and Cell Membranes 74
I. Introduction 74
II. Experimental Approaches 75
III. The Common Features of Electroporation 75
IV. Mechanisms of Lipid Bilayer Electroporation 77
V. The Specificity of Electroporation for Biomembranes 79
VI. Pores and Electrotransfection of Cells 81
Acknowledgments 84
References 84
Chapter 6. Biophysical Considerations of Membrane Electroporation 88
I. Introduction 88
II. Electrooptic and Conductometric Relaxations of Lipid Bilayer Vesicles 89
III. Electroporation and Membrane Permeability 93
IV. Electroporative DISIA Transfer 99
Acknowledgments 101
References 101
Chapter 7. Progress toward a Theoretical Model for Electroporation Mechanism: Membrane Electrical Behavior and Molecular Transport 102
I. Introduction 103
II. Theory of Electroporation 104
III. Molecular Transport 115
IV. Isolated Cell Electroporation Model 122
V. Related Experimental Issues 123
Acknowledgments 125
References 125
Chapter 8. Mechanisms of Electroporation and Electrofusion 130
I. Introduction and Scope 130
II. Electroosmosis in Electropores 131
III. Electrofusion and Electroporation Protocols 136
IV. Criteria for Membrane Fusion 136
V. New Fusion Product Understanding and Its Implications for Electrofusion 138
VI. Factors That Influence Electrofusion 140
VII. Misconceptions 144
Acknowledgment 144
References 144
Chapter 9. Interfacfal Membrane Alteration Associated with Electropermeabilization and Electrofusion 150
I. Introduction 150
II. Electropermeabilization 151
III. Permeabilization Quantification 152
IV. Time Course of Events Associated with Electropermeabilization 153
V. Electropermeabilized Cells Are Fusogenic 158
VI. Molecular Alterations of the Cell Membrane during Electropermeabilization 159
VII. Chemical and Physical Alterations of the Membrane Affect the Expansion Step 160
VIII. Conclusions 162
Acknowledgments 162
References 163
Chapter 10. Membrane Fusion Kinetics 166
I. Introduction 166
II. Fusion Kinetics 167
III. Delays 169
IV. Rates of Fusion 171
V. Fusion Yields 172
VI. Models of Fusion Kinetics 173
VII. Conclusion 175
Acknowledgments 175
References 175
Chapter 11. Effects of Intercellular Forces on Electrofusion 178
I. Introduction 178
II. Theory 179
III. Calculation of the Pulse-Induced Pressure 180
IV. Experimental Verification 185
V. Concluding Remarks 188
Acknowledgment 188
References 188
Chapter 12. Dynamics of Cytoskeletal Reorganization in CV-1 Cells during Electrofusion 190
I. Introduction 190
II. Materials and Methods 192
III. Results 195
IV. Discussion 204
V. Conclusion 207
Acknowledgments 207
References 207
Part II: Applications of Electroporation and Electrofusion in Current Research 210
Chapter 13. Gene Transfer into Adherent Cells Growing on Microbeads 212
I. Introduction 212
II. Advantages of Electroporation for Gene Transfection 213
III. Applications of Electroporation in Molecular and Cellular Biology 214
IV. Potential Applications to Human Gene Therapy 215
V. Electroporation of Adherent Cells on Microbeads 215
Acknowledgments 218
References 218
Chapter 14. Gene Targeting and Electroporation 220
I. Introduction 220
Il. Types of Gene Targeting Constructs 221
III. Methods of Isolating Targeted Cells 221
IV. Transferring Targeted Alterations to the Mouse Germ Line 231
V. DNA Integration in Targeted Cells 232
VI. Conclusion 233
Acknowledgments 233
References 233
Chapter 15. Pollen Electrotransformation for Gene Transfer in Plants 238
I. Introduction 238
II. Current Research on Electroporation 240
III. Previous Reports on Pollen Transformation 241
IV. Pollen Biology 242
V. Electrotransformation of Pollen 243
VI. Analysis of Transformed Plants 252
VII. Summary 254
Acknowledgments 254
References 254
Chapter 16. Electrofusion of Plant Protoplasts and the Production of Somatic Hybrids 260
I. Introduction 260
II. Factors Affecting Protoplast Electrofusion 261
III. Somatic Hybridization by Electrofusion 263
IV. Comparison of Electrofusion with PEG-lnduced Fusion 270
References 271
Chapter 17. Electrotransformation of Bacteria by Plasmid DNA 276
I. Development of Bacterial Electrotransformation 276
II. The Application of Electroporation to Bacteria 282
III. Factors Affecting Electrotransformation 285
Acknowledgments 294
References 294
Chapter 18. Creating Vast Peptide Expression Libraries: Electroporation as a Tool to Construct Plasmid Libraries of Greater than 109 Recombinants 302
I. Introduction 302
II. Electroporation as a Tool to Create Extremely Large Libraries 303
III. Constructing Peptide Expression Libraries: An Application Exploiting the High DNA Capacity of Electrotransformation 307
IV. Analysis of the Libraries 309
V. Conclusions 311
References 312
Chapter 19. Electroporation and Electrofusion Using a Pulsed Radio-Frequency Electric Field 314
I. Introduction 314
II. Materials and Methods 317
III. Results 321
IV. Discussion 335
References 336
Chapter 20. Electroinsertion: An Electrical Method for Protein Implantation into Cell Membranes 338
I. Introduction 339
II. Some Key Factors of Electroinsertion 340
III. Procedures 342
IV. Detection of Electroinserted Proteins 345
V. Orientation of the Electroinserted Proteins 351
VI. Life-Span Study 353
VII. Electroinsertion in Nucleated Cell Plasma Membrane 354
VIII. Conclusions 354
References 354
Chapter 21. Electroporation as a Tool to Study Enzyme Activities in Situ 358
I. Introduction 358
III. Experimental Considerations 359
III. Permeabilized Cell Assays 362
IV. Conclusions 371
References 371
Chapter 22. Comparison of PEG-lnduced and Electric Field-Mediated Cell Fusion in the Generation of Monoclonal Antibodies against a Variety of Soluble and Cellular Antigens 374
I. Introduction 374
II. Electrofusion as Presently Used in Berlin-Buch 375
III. Results 377
IV. Conclusions 379
References 379
Chapter 23. Production of Genetically Identical Embryos by Electrofusion 382
I. Introduction 382
II. The Amphibian Cloning Model 383
III. The Mammalian Cloning Model 385
IV. IMuclear–Cytoplasmic Interactions 394
V. Applications and Conclusions 396
Acknowledgments 398
References 398
Chapter 24. Development of Cell–Tissue Electrofusion for Biological Applications 404
I. Introduction 405
II. Biological Applications 405
III. In Vitro Cell–Tissue Electrofusion 409
IV. In Situ and in Vivo Cell-Tissue Electrofusion 412
V. Quantitative Assay for Cell–Tissue Electrofusion 418
VI. Standardized Reproducible Cell–Tissue Electrofusion 418
VII. Summary 419
References 419
Chapter 25. Novel Applications of Electroporation 422
I. Introduction 422
II. Protein Loading 423
III. Virus 427
IV. Whole Tissue 429
V. Transgenic Fish 430
VI. Cell Monolayers 431
VII. Improved Cell Viability and Transformation Efficiency 432
References 433
Part III: Practical Protocols for Electroporation and Electrofusion 438
Chapter 26. Design of Protocolsfor Electroporation and Electrofusion: Selection of Electrical Parameters 440
I. Introduction 441
II. Electroporation Using a DC Field 443
III. Electrofusion Using a DC Field 453
IV. Electroporation and Electrofusion Using a Radiofrequency Field 457
Acknowledgments 460
References 460
Chapter 27. Protocols for Using Electroporation to Stably or Transiently Transfect Mammalian Cells 468
I. Introduction 468
II. Materials 469
III. Electroporation Parameters 470
IV. Procedures 470
Acknowledgments 473
References 474
Chapter 28. Optimization of Electroporation Using Reporter Genes 476
I. Introduction 476
II. Materials 479
III. Methods 480
Acknowledgment 481
References 481
Chapter 29. Genetic Manipulation of Plant Cellsby Means of Electroporation and Electrofusion 482
I. Introduction 482
II. Transformation by Electroporation 486
III. Electrofusion 488
IV. Use of Oscilloscope for Pulse Analysis 491
V. Selection 491
VI. Conclusion 493
References 493
Chapter 30. Protocols for the Transformation of Bacteria by Electroporation 496
I. Introduction 496
II. General Protocol for the Electrotransformation of Gram-Negative Species 497
III. The Electrotransformation of Gram-Positive Species 501
References 508
Chapter 31. Protocol for High-Efficiency Yeast Transformation 512
I. Introduction 512
II. Materials 513
III. Procedures 514
IV. Modifications 515
Acknowledgments 515
References 515
Chapter 32. Protocols of Electroporation and Electrofusion for Producing Human Hybridomas 518
I. Introduction 518
II. Materials 521
III. Procedures 522
IV. Summary 530
References 530
Chapter 33. Human Hybridoma Formation by Hypo-Osmolar Electrofusion 534
I. Introduction 534
II. Materials 536
III. Procedure 538
Acknowledgments 543
References 543
Chapter 34. Electrically Induced Fusion and Activation in Nuclear Transplant Embryos 546
I. Introduction 546
II. Materials 549
III. Procedures 550
IV. Discussion 554
V. Conclusion 560
Acknowledgments 560
References 560
Part IV: Instrumentation for Electroporation and Electrofusion 564
Chapter 35. Pulse Generators for Electrofusion and Electroporation 566
I. Introduction 566
II. Commercially Available Generators Designed for Electrofusion and Electroporation 568
III. Homemade and Commercial Pulse Generators 577
References 579
Index 582
Overview of Electroporation and Electrofusion
Donald C. Chang1 1 Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, Texas 77030
Present address: Department of Biology, Hong Kong University of Science and Technology, Kowloon, Hong Kong.
James A. Saunders2 2 Plant Sciences Institute, Beltsville Agricultural Research Center, United States Department of Agriculture, Beltsville, Maryland 20705
Bruce M. Chassy3 3 Department of Food Science, University of Illinois, Urbana, Illinois 61801
Arthur E. Sowers4 4 Department of Biophysics, School of Medicine, University of Maryland at Baltimore,Baltimore, Maryland 21201
II. Advantages of Electroporation and Electrofusion
III. Mechanisms of Electroporation and Electrofusion
IV. Applications of Electroporation
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.
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 externally 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 Rosenheck, 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 molecules, 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 manipulation, (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).
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 (Scheurich 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 beginning 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 μs, or it can be an exponentially decaying capacitive discharge pulse with a duration in the millisecond range (Saunders et al., 1989).
When the induced potential reaches a critical value, it causes an electrical breakdown 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 investigation at this time (See Part I). The mechanisms by which membranes of neighboring 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 irreversible 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, transformation 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, electrotransfection, 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...
| Erscheint lt. Verlag | 2.12.2012 |
|---|---|
| Sprache | englisch |
| Themenwelt | Naturwissenschaften ► Biologie ► Genetik / Molekularbiologie |
| Naturwissenschaften ► Biologie ► Zellbiologie | |
| Technik | |
| ISBN-10 | 0-08-091727-5 / 0080917275 |
| ISBN-13 | 978-0-08-091727-6 / 9780080917276 |
| Haben Sie eine Frage zum Produkt? |
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