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Plant Cell Electroporation and Electrofusion Protocols
Methods in Molecular BiologyTM Series John M. Walker, SERIES EDITOR
55. PUnt Cell Electroporation and Electrofusion Protocols, edited by Jac A. Nickoloff. 1995
54. ¥AC Protocols, edited by David Morfec, 1995
53. Yeast Protocols: Methods in Cell and Molecular Biology,
edited by Ivor H. Evans, 1995
52. Capillary Electrophoresis: Principles, Instrumentation,
and Applications, edited by Kevin D. Attria, 1995
51. Antibody Engineering Protocols, edited by Sudhir Paul,
1995
50. Species Diagnostics Protocols: PCR and Other Nucleic
Acid Methods, edited by Justin P. Clapp. 1995 49. Plant Gene Transfer and Expression Protocols,
edited by Heddwyn Jones. 1995 48. Animal Cell Electroporation and Electrofusion Proto
cols, edited by Jac A. Nickoloff, 1995 47. Electroporation Protocols for Microorganisms, edited by
Jac A. Nickoloff, 1995 46. Diagnostic Bacteriology Protocols, edited by Jenny
Howard and David M. Whitcombe, 1995 45. Monoclonal Antibody Protocols, edited by William C.
Davis, 1995
44. AgrobacteriuM Protocols, edited by Kevan M. A. Cartland
and Michael R. Davey, 1995
43. In Vitro Toxicity Testing Protocols, edited by Sheila
O'Hare and Chris K. Alterwill, 1995
42. ELISA: Theory and Practice, by John R. Crowlher, 1995
41. Signal Transduction Protocols, edited by David A. Kendall
and Stephen J. Hill, 1995
40. Protein Stability and Folding: Theory and Practice,
edited by Bret A. Shirley, 1995
39. Baculovirus Expression Protocols, edited by Christopher D. Richardson, 1995
35. Cryopreservation and Freeze-Drying Protocols, edited by John G. Day and Mark R. McLellan, 1995
37. In Vitro Transcription and Translation Protocols, edited by Martin J. Tymms, 1995
36. Peptide Analysis Protocols, edited by Ben M. Dunn and Michael W. Pennington, 1994
35. Peptide Syntliesis Protocols, edited by Michael W. Pennington and Ben M. Dunn. 1994
34. Immunocytochemical Methods and Protocols, edited by
Lorette C. Javois, 1994
33. In Silu Hybridization Protocols, edited by K. H. Andy
Choo, 1994
32. Basic Protein and Peptide Protocols, edited by John M.
Walker, 1994
31. Protocols for Gene Analysis, edited by Adrian J. Harwood,
1994
30. DNA-Protein Interactions, edited by G. GeoffKneale, 1994
29. Chromosome Analysis Protocols, edited by John R. Gosden, 1994
28. Protocols for Nucleic Acid Analysis by Nonradioactive Probes, edited by Peter G. Isaac, 1994
27. Biomembrane Protocols: //. Architecture and Function, edited by John M. Graham and Joan A. Higgins, 1994
26. Protocols for Oligonucleotide Conjugates: Synthesis and Analytical Techniques, edited by Sudhir Agrawal, 1994
25. Computer Analysis of Sequence Data: Part //, edited by Annette M. Griffin and Hugh G. Griffin. 1994
24. Computer Analysis of Sequence Data: Part I. edited by Annette M. Griffin and Hugh G. Griffin, 1994
23. DNA Sequencing Protocols, edited by Hugh G. Griffin and Annette M. Griffin, 1993
22. Microscopy, Optical Spectroscopy, and Macroscopic Techniques, edited by Christopher Jones, Barbara MuUoy, and Adrian H. Thomas, 199S
21. Protocols in Molecular Parasitology, edited by John E. Hyde. 1993
20. Protocols for Oligonucleotides and Analogs: Synthesis and Properties, edited by Sudhir Agrawal, 1993
19. Biomembrane Protocols: /. Isolation and Analysis. edited by John M. Graham and Joan A. Higgins, 1993
18. Transgenesis Techniques: Principles and Protocols, edited by David Murphy and David A. Carter, 1993
17. Spectroscopic Methods and Analyses: NMR, Mass Spectrometry, and Metalloprotein Techniques, edited by Christopher Jones, Barbara Mulloy, and Adrian H. Thomas, 1993
16. Enzymes of Molecular Biology, edited by Michael M. Barren, 1993
15. PCR Protocols: Current Methods and Applications, edited by Bruce A. White, 1993
14. Glycoprotein Analysis in Biomedicine, edited by Elizabeth F. Hounsell, 1993
13. Protocols in Molecular Neurobiology, edited by Alan Longstaffand Patricia Revest, 1992
12. Pulsed-Field Gel Electrophoresis: Protocols, Methods, and Theories, edited by Margii Burmeisler and Levy Ulanovsky, 1992
11. Practical Protein Chromatography, edited by Andrew Kenney and Susan Fowell, 1992
10. Immunochemical Protocols, edited by Margaret M. Manson. 1992
9. Protocols in Human Molecular Genetics, edited by Christopher G. Mathew, 1991
8. Practical Molecular Virology: Viral Vectors for Gene Expression, edited by Mary K. L Collins, 1991
7. Gene Transfer and Expression Protocols, edited by Edward J. Murray, 1991
6. Plant Cell and Tissue Culture, edited by Jeffrey W. Pollard and John M. Walker, 1990
5. Animal Cell Culture, edited by ycj(/"re>' W. Pollard and John M. Walker, 1990
Methods in Molecular Biology™ • 55
Plant Cell Electroporation
and Electrofusion Protocols
Edited by
Jac A. Nickoloff Harvard University, Boston, MA
H u m a n a P r e s s ^ j ^ Totowa, New Jersey
© 1995 Humana Press Inc. 999 Riverview Drive, Suite 208 Totowa, New Jersey 07512
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Library of Congress Cataloging-in-Publication Data
Plant cell electroporation and electrofusion protocols/edited by Jac A. Nickoloff. p. cm—{Methods in molecular biology'";55)
Includes indexes. ISBN 0-89603-328-7 (alk. paper) 1. Plant genetic engineering—Laboratory manuals. 2. Plant genetic transformation—Laboratory
manuals. 3. Crops—Genetic engineering—Laboratory manuals. 4. Electroporation—Laboratory manuals. 5. Electrofusion—Laboratory manuals. I. Nickoloff, Jac A. II. Series: Methods in molecular biology™ (Totowa, NJ); 55. QK981.5.P55 1995 581.87'328—dc20 95-358
CIP
Preface
Gene transfer is an essential technology for improving our understanding of gene structure and function. Although there are many methods by which DNA may be introduced into cells—including heat and chemical treatments, and microinjection—electroporation has been found to be the most versatile gene transfer technique. Electroporation is effective with a wide variety of cell types, including those that are difficult to transform by other means. For many cell types, electroporation is either the most efficient or the only means known to effect gene transfer. The early and broad success of electric field-mediated DNA transfer soon prompted researchers to investigate electroporation for transferring other types of molecules into cells, including RNA, enzymes, antibodies, and analytic dyes.
The first section of Plant Cell Electroporation and Electrofusion Protocols includes two chapters that serve as a guide to theoretical and practical aspects of electroporation, and will be of particular interest to those developing protocols for as yet untested species or cell types, and a third chapter that describes commercially available electroporation instruments. The remaining chapters describe well-tested protocols for DNA electrotransfection, electroporation of other biomolecules, or cell electrofusion. These chapters also include brief discussions of alternatives to electric field-based methods, citing the advantages and limitations of the various methods for achieving specific goals. Electroporation has become a favored method for introducing DNA into, and fusing plant cells, and as such these technologies promise to play a pivotal role in the development of more productive, hardy crop plants, and of plants resistant to insects and microbial pathogens. Of particular interest is the recently developed method for pollen electrotransformation, since this
vi Preface
holds significant promise for making stably inherited changes in plants by direct modification of the germ line. Technologies for transforming plant cells lagged behind those for transforming microorganisms and animal cells. The advent of plant cell electrotransformation methods has had a strong impact on both basic and applied plant research.
Although electroporation procedures for different cell types are often similar, subtle differences in either electrical parameters or growth conditions can have strong effects on transfection or fusion efficiencies, and such factors are often important to control when optimum efficiencies are required. Each protocol, therefore, provides considerable detail about conditions for growing and preparing cell and tissue samples, with many helpful troubleshooting tips. Although widely used, electroporation is still a relatively young technology, and it is expected that this collection of protocols will both propel the electric field technologies forward and facilitate the growth in our understanding of the biological processes that these technologies are used to explore.
I want to thank all of the contributors for their timely and high quality submissions, with special thanks to Dr. Patrick Gallois for his many reconunendations of potential contributors. I also want to thank series editor Dr. John Walker for his valuable assistance and advice during the editing process.
Jac A. Nickoloff
Contents
Preface v
Contents for the Companion Volumes ix
Contributors xiii
PART I. THEORY AND INSTRUMENTATION 1
CH. 1. Electioporation Theory: Concepts and Mechanisms, James C. Weaver 3
CH. 2. Effects of Pulse Length and Strength on Electroporation Efficiency, Sek Wen Hui 29
CH. 3. Instrumentation, GunterA. Hofmann 41
PART II. ELECTROPORATION PROTOCOLS 61
CH. 4. ElectiopoTation of Agrobacterium tumefaciens, Amke den Dulk-Ras and PaulJ. J. Hooykaas 63
CH. 5. Electroporation of DNA into the Unicellular Green Alga Chlamydomonas reinhardtii,
Laura R. Keller 73 CH. 6. Pollen Electrotransformation in Tobacco,
James A. Saunders and Benjamin F. Matthews 81 CH. 7. Electroporation of Tobacco Leaf Protoplasts Using Plasmid DNA
or Total Genomic DNA, Patrick Gallois, Keith Lindsey, and Renee Malone 89
CH. 8. Electroporation of firajsica, Frank Siegemund and Klaus Eimert 709
CH. 9. Transformation of Maize by Electroporation of Embryos, Carol A. Rhodes, Kathleen A. Marrs, and Lynn E. Murry 121
CH. 10. Transient Gene Expression Analysis in Electroporated Maize Protoplasts,
Kathleen A. Marrs and J. C. Carle Urioste 133 CH. 11. Reporter Genes and Transient Assays for Plants,
Benjamin F. Matthews, James A. Saunders, Joan S. Gebhardt, Jhy-Jhu Lin, and Susan M. Koehler 147
Vll
viii Contents
PART in. ELECTROFUSION PROTOCOLS 163
CH. 12. Electrofusion of Plant Protoplasts: Selection and Screening for Somatic Hybrids o/Nicotiana,
Harold N. Trick and George W. Bates 165 CH. 13. Protoplast Electrofusion and Regeneration in Potato,
Jianping Cheng and James A. Saunders 181 CH. 14. Polymer-Supported Electrofusion of Protoplasts: A Novel Method
and a Synergistic Effect, Lei Zhang 189
Index 203
CONTENTS FOR THE COMPANION VOLUME
Electroporation Protocols for Microorganisms
CH. 1. Electroporation TheoTy: Concepts and Mechanisms, James C. Weaver
CH. 2. Instrumentation, GunterA. Hofmann
CH. 3. Direct Plasmid Transfer Between Bacterial Species and Electrocuring, Helen L. Withers
CH. 4. Transfer of Episomal and Integrated Plasmids from Saccharomyces cerevisiae to Escherichia coli by Electroporation,
Laura Gunn, Jennifer Whelden, and Jac A. Nickoloff CH. 5. Production of cDNA Libraries by Electroporation,
Christian E. Gruber CH. 6. Electroporation of RNA into Saccharomyces cerevisiae,
Daniel R. Gallie CH. 7. Electrofusion of Yeast Protoplasts,
Herbert Weber and Hermann Berg CH. 8. Escherichia coli Electrotransformation,
Elizabeth M. Miller and Jac A. Nickoloff CH. 9. Electrotransformation in Salmonella,
Kenneth E. Sanderson, P. Ronald MacLachlan, and Andrew Hessel CH. 10. Electrotransformation of PjcMcfomonaj,
Jonathan J. Dennis and Pamela A. Sokol CH. 11. Electroporation of Xanthomonas,
Teresa J. White and Carlos F. Gonzalez CH. 12. Transformation of Brucella Species with Suicide and Broad Host-Range
Plasmids, John R. McQuiston, Gerhardt G. Schurig, Nammalwar Srirangathan,
and Stephen M. Boyle CH. 13. Electroporation of Francisella tularenis,
Gerald S. Baron, Svetlana V. Myltseva, and Francis E. Nano CH. 14. A Simple and Rapid Method for Transformation of Vibrio Species
by Electroporation, Hajime Hamashima, Makoto Iwasaki, and Taketoshi Aral
CH. 15. Genetic Transformation of Bacteroides spp. Using Electroporation, C. Jeffrey Smith
CH. 16. Electrotransformation of AgrofcacferiMm, Jhy-Jhu Lin
IX
X Contents for Companion Volumes
CH. 17. ElectiopoTationof Helicobacter pylori, Ellyn D. Segal
CH. 18. Electrotransformation of Sfrepfococci, Robert E. McLaughlin and Joseph J. Ferretti
CH. 19. Transformation of Lactococcus by Electroporation, Helge Holo and IngolfF. Nes
CH. 20. Transformation of Lactobacillus by Electroporation, Thea W. Aukrust, May B. Brurberg, and IngolfF. Nes
CH. 21. Electrotransformation of Staphlococci, Jean C. Lee
CH. 22. Electroporation and Efficient Transformation of Enterococcus faecalis Grown in High Concentrations of Glycine,
Brett D. Shepard and Michael S. Gilmore CH. 23. Introduction of Recombinant DNA into Clostridium spp.,
Mary K. Phillips-Jones CH. 24. Electroporation of Mycobacteria,
T. Parish andN. G. Stoker CH. 25. Electrotransformation of the Spirochete Borrelia burgdorferi,
D. Scott Samuels CH. 26. Yeast Transformation and the Preparation of Frozen Spheroplasts
for Electroporation, Lisa Stowers, James Gautsch, Richard Dana, and Merl F. Hoekstra
CH. 27. Ten-Minute Electrotransformation of Saccharomyces cerevisiae, Martin Grey and Martin Brendel
CH. 28. Electroporation of Schizosaccharomyces pombe, Mark T. Hood and C. 5. Stachow
CH. 29. Gene Transfer by Electroporation of Filamentous Fungi, M. Kapoor
CH. 30. Transformation of Candida maltosa by Electroporation, Dietmar Becher and Stephen G. Oliver
CH. 31. Electroporation of Physarum polycephalum, Timothy G. Burland and Juliet Bailey
CH. 32. Electroporation of Dictyostelium discoideum, David Knecht and K. Ming Pang
CH. 33. Gene Transfer by Electroporation of Tetrahymena, Jacek Gaertig and Martin A. Gorovsky
CH. 34. Transfection of the African and American trypanosomes, John M. Kelly, Martin C. Taylor, Gloria Rudenko,
and Pat A. Blundell CH. 35. Electroporation in Giardia lamblia,
A. L. Wang, Tiina Sepp, and C. C. Wang Index
PART I.
CH.
CH.
CH.
1.
2.
3.
PART H,
CH. 4.
CONTENTS FOR THE COMPANION VOLUME
Animal Cell Electroporation and Electrofusion Protocols
THEORY AND INSTRUMENTATION
Electroporation Theory: Concepts and Mechanisms, James C. Weaver
Effects of Pulse Length and Strength on Electroporation Efficiency, Sek Wen Hui
Instrumentation, GunterA. Hofmann
ELECTROPORATION PROTOCOLS
The Introduction of Proteins into Mammalian Cells by Electroporation, William F. Morgan and Joseph P. Day
CH. 5. Electroporation of Antigen-Presenting Cells for T-Cell Recognition and Cytotoxic T-Lymphocyte Priming,
Weisan Chen and James McCluskey CH. 6. Electroporation of Antibodies into Mammalian Cells,
Paul L. Campbell, James McCluskey, Jing Ping Yeo, and Ban-Hock Toh
CH. 7. Electroporation of Adherent Cells In Situ for the Introduction of Nonpermeant Molecules
Leda H. Raptis, Kevin L. Firth, Heather L. Brownell, Andrea Todd, W. Craig Simon, Brian M. Bennett, Leslie W. MacKenzie, and Maria Zannis-Hadjopoulos
CH. 8. Eletrotransformation of Chinese Hamster Ovary Cells, Danielle Gioioso Taghian and Jac A. Nickoloff
CH. 9. Eletroporation of Rat Pituitary Cells, Ruth H. Paulssen, EyvindJ. Paulssen, and Kaare M. Gautvik
CH. 10. Electroporation of Plasmid DNA into Normal Human Fibroblasts, F. Andrew Ray
CH. 11. Electroporation-Mediated Gene Transfer into Hepatocytes, Alphonse Le Cam
CH. 12. Electroporation of Human Lymphoblastoid Cells, Fen Xia and Howard L. Liber
CH. 13. The Use of Electroporated Bovine Spermatozoa to Transfer Foreign DNA into Oocytes,
Marc Gagne, Frangois Pothier, and Marc-Andre Sirard
xi
xii Contents for Companion Volumes
CH. 14. Electroporation of Embryonic Stem Cells for Generating Transgenic Mice and Studying In Vitro Differentiation,
John S. Mudgett and Thomas J. Livelli CH. 15. Electrotransfection with "Intracellular" Buffer,
Maurice J. B. van den Hoff, Vincent M. Christoffels, Wil T. Labruyere, Antoon F. M. Moorman, and Wouter H. Lamers
CH. 16. Effect of Cis-Located Human Satellite DNA on Electroporation Efficiency,
Djennan Saint-Die and Michael S. DuBow CH. 17. Quantitation of Trasient Gene Expression,
Michael K. Showe and Louise C. Showe CH. 18. Stable Integration of Vectors at High Copy Number for High-Level
Expression in Animal Cells, James Barsoum
CH. 19. Electroporation of Drosophilia Embryos, K. Puloma Kamdar, Thao N. Wagner, and Victoria Finnerty
CH. 20. Transformation of Fish Cells and Embryos, Koji Inoue, Jun-ichiro Hata, and Shinya Yamashita
CH. 21. Electroporation of Cardiac Cells, Leslie Tung
CH. 22. Electroporation for Gene Therapy, Kathryn Matthews, Sukhendu B. Dev, Frances Toneguzzo,
and Armand Keating
PART III. ELECTROFUSION PROTOCOLS
CH. 23. Electrofusion of Mammalian Cells, Kenneth L. White
CH. 24. Stabilizing Antibody Secretion of Human Epstein Barr Virus-Activated B-Lymphocytes with Hybridoma Formation by Electrofusion,
Susan Perkins and Steven K. H. Foung CH. 25. Electrofusion of Mammalian Oocytes and Embryonic Cells,
Josef Fulka, Jr., Robert M. Moor, and Josef Fulka CH. 26. Nuclear Transfer in Bovine Embryos,
Akira Iritani and Tasuku Mitani CH. 27. Electrofusion of Mouse Embryos to Produce Tetraploids,
Ulrich Petzoldt CH. 28. Spectrofluorometric Assay for Cell-Tissue Electrofusion,
Richard Heller CH. 29. Cytometric Detection and Quantitation of Cell-Cell Electrofusion
Products, Mark J. Jaroszeski, Richard Gilbert, and Richard Heller
Index
Contributors
GEORGE W . BATES • Department of Biological Science, Florida State University, Tallahassee, FL
JiANPiNG CHENG • Plant Sciences Institute, USDA, ARS, Beltsville, MD AMKE DEN DULK-RAS • Institute of Molecular Plant Sciences, Clusius
Laboratory,. Leiden University, Leiden, The Netherlands KLAUS EIMERT • Department of Genetics, University of Halle, Saale,
Germany PATRICK GALLOIS • Laboratoire de Physiologie et Biologie
Moleculaire Vegetale, Universite de Perpignan, France JOAN S. GEBHARDT • Plant Molecular Biology Laboratory, USDA-ARS,
Beltsville, MD GUNTER A . HOFMANN • Genetronics Inc., San Diego, CA PAUL J. J. HOOYKAAS • Institute of Molecular Plant Sciences, Clusius
Laboratory, Leiden University, Leiden, The Netherlands SEK WEN HUI • Membrane Biophysics Laboratory, Biophysics
Department, Roswell Park Cancer Institute, Buffalo, NY LAURA R. KELLER • Department of Biological Science, Florida State
University, Tallahassee, FL SUSAN M . KOEHLER • USDA, APHIS, BBEP, Hyattsville, MD JHY-JHU LIN • Life Technologies, Gaithersburg, MD KEITH LINDSEY • Depatment of Botany, University of Leicester, UK RENEE MALONE • Waksman Institute, Rutgers University, Piscataway,
NJ KATHLEEN A. MARRS • Department of Biological Sciences, Stanford
University, Stanford, CA BENJAMIN F . MATTHEWS • Plant Molecular Biology Laboratory,
USDA-ARS, Beltsville, MD LYNN E . MURRY • Sandoz Agro, Inc., Palo Alto, CA CAROL A. RHODES • Sandoz Agro, Inc., Palo Alto, CA
xiii
xiv Contributors
JAMES A. SAUNDERS • Soybean and Alfalfa Research Laboratory, Plant Sciences Institute, USDA-ARS, Beltsville, MD
FRANK SIEGEMUND • Department of Genetics, University of Halle, Saale, Germany
HAROLD N . TRICK • Department of Biological Sciences, Florida State University, Tallahassee, FL
J. C. CARLE URIOSTE • Department of Biological Sciences, Stanford University, Stanford, CA
JAMES C. WEAVER • Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA
LEI ZHANG • Laboratory of Bioelectrochemistry, Institute of Molecular Biotechnology, Jena, Germany. Present Address: Department of Biology, Massachusetts Institute of Technology, Cambridge, MA