Guide To 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. 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

Cells can be funny. Try to grow them with a slightly wrong recipe, and they turn over and die. But hit them with an electric field strong enough to knock over a horse, and they do enough things to justify international meetings, to fill a sizable book, and to lead one to speak of an entirely new technology for cell manipulation. The very improbability of these events not only raises questions about why things happen but also leads to a long list of practical systems in which the application of strong electric fields might enable the merger of cell contents or the introduction of alien but vital material. Inevitably, the basic questions and the practical applications will not keep in step. The questions are intrinsically tough. It is hard enough to analyze the action of the relatively weak fields that rotate or align cells, but it is nearly impossible to predict responses to the cell-shredding bursts of electricity that cause them to fuse or to open up to very large molecular assemblies. Even so, theoretical studies and systematic examination of model systems have produced some creditable results, ideas which should ultimately provide hints of what to try next.

Gene transfer is an essential technology for improving our under standing of gene structure and function. Although there are many meth ods 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-medi ated DNA transfer soon prompted researchers to investigate electroporation for transferring other types of molecules into cells, in cluding 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 alterna tives to electric field-based methods, citing the advantages and limita tions of the various methods for achieving specific goals.

The ability to introduce macromolecules into animal cells, includ ing DNA, RNA, proteins, and other bioactive compounds has facili tated a broad range of biological studies, from biochemistry and biophysics to molecular biology, cell biology, and whole animal stud ies. Gene transfer technology in particular will continue to play an essential role in studies aimed at improving our understanding of the relationships between the gene structure and function, and it has impor tant practical applications in both biotechnology and biomedicine, as evidenced by the current intense interest in gene therapy. Although DNA and other macromolecules may be introduced into cells by a variety of methods, including chemical treatments and microinjection, el- troporation has proven to be simpler to perform, more efficient, and effective with a wider variety of cell types than other techniques. 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. Animal Cell Electroporation and Electrofusion Protocols begins with three chapters that describe the theoretical and practical aspects of electroporation, including a review of the commercially available instrumentation. These introductory chapters will be of particular inter est to those new to electric field technologies and to those developing protocols for as yet untested species or cell types. Nineteen chapters follow that present well-tested protocols for electroporation of proteins and DNA into insect, fish, and mammalian cells.

This book presents a comprehensive and coherent picture of how molecules diffuse across a liquid that is, on average, only two molecules thick. It begins by characterizing bilayers structurally, using X-ray diffraction, and then mechanically by measuring elastic moduli and mechanisms of failure. Emphasis is placed on the stability and mechanical properties of plant membranes that are subject to very large osmotic and thermal stresses. Using this information, the transport of molecules of increasing complexity across bilayers is analyzed.

This volume is volume entirely dedicated to microfabricated cell-based systems. It will provide readers with a quick introduction to the field as well as with a variety of specific examples of such Lab-on-Chip systems for cellomics applications. It will give investigators inspiration for innovative research topics, whereas end users will be surprised about the wide variety of new and exciting applications.

The use of fluorescent and luminescent probes to measure biological function has increased dramatically since publication of the First Edition due to their improved speed, safety, and power of analytical approach. This eagerly awaited Second Edition, also edited by Bill Mason, contains 19 new chapters and over two thirds new material, and is a must for all life scientists using optical probes. The contents include discussion of new optical methodologies for detection of proteins, DNA and other molecules, as well as probes for ions, receptors, cellular components, and gene expression. Emerging and advanced technologies for probe detection such as confocal laser scanning microscopy are also covered. This book will be essential for those embarking on work in the field or using new methods to enhance their research. TOPICS COVERED: * Single and multiphoton confocal microscopy * Applications of green fluorescent protein and chemiluminescent reporters to gene expression studies * Applications of new optical probes for imaging proteins in gels * Probes and detection technologies for imaging membrane potential in live cells * Use of optical probes to detect microorganisms * Raman and confocal raman microspectroscopy * Fluorescence lifetime imaging microscopy * Digital CCD cameras and their application in biological microscopy

This book is a comprehensive reference on the methods available for the enhancement of percutaneous penetration. It examines a broad scope of chemical enhancers and various physical methods of enhancement. The range of chemicals discussed in this volume is unsurpassed anywhere in the literature. Scientists can find invaluable information in this single source on 95 percent of the enhancers being researched today! Each well-illustrated chapter is written by the world's leading authorities in the field. Introductory chapters outline the concept of penetration enhancement from first principles and provide a comprehensive classification of different types of enhancers.Chapters covering chemical enhancers are clear, concise, and easy to understand, even for novice readers in this field. The latest techniques by which drugs can be forced physically through the skin are also covered in depth. Equipment is detailed, typical drug candidates for the techniques are described, and the success to date is provided. Percutaneous Penetration Enhancers is the first book to present modern analytical techniques that have been used to assess penetration enhancement. The book also offers future perspectives for enhancers. New approaches and current limitations on knowledge and understanding of the topic are suggested. This timely source book is invaluable for clinical reference or for the design of scientific studies.