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

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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

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 Biology™ is a trademark of The Humana Press Inc.

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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