Gene Transfer To Animal Cells

Gene transfer to animal cells was first achieved more than thirty years ago. Since then, transformation technology has developed rapidly, resulting in a multitude of techniques for cell transformation and the creation of transgenic animals. As with any expanding technology, it becomes difficult to keep track of all the developments and to find a concise and comprehensive source of information that explains all the underlying principles. Gene Transfer to Animals Cells addresses this problem by describing the principles behind gene transfer technologies, how gene expression is controlled in animal cells and how advanced strategies can be used to add, exchange or delete sequences from animal genomes in a conditional manner. A final chapter provides an overview of all the applications of animal cell transformation in farming, medicine and research.

This volume provides a broad, state-of-the-art coverage of diverse technical topics in gene expression in mammalian cells, including the development of vectors for production of proteins in cultured cells, in transgenic animals, vaccination, and gene therapy; progress in methods for the transfer of genes into mammalian cells and the optimization and monitoring of gene expression; advances in our understanding and manipulation of cellular biochemical pathways that have a quantitative and qualitative impact on mammalian gene expression; and the large-scale production and purification of proteins from cultured cells.

This book includes the proceedings of a NATO Advanced Study Institute held at Washington State University, Pullman, Washington from July 26 until August 6, 1982. Although genetic engineering in eukaryotes is best developed in yeast and mammalian cells, the reader will find that some emphasis has been put on plant systems. Indeed, it was our position that the development of plant cell genetic transformation would benefit from the interactions between a comparatively smaller number of fungal and animal cell experts and a larger number of plant cell specialists representing various aspects of plant molecular genetic research. On the other hand, it is clear that the ultimate achievements of plant genetic engineering will have a tremendous ~pact on, among other things, food production without generating the problems of ethics encountered when one contempla-tes the genetic modification of human beings. Therefore, this slight bias in favor of the plant kingdom simply reflects our belief that a "second green revolution" will benefit mankind to a greater extent than any other kind of genetic engine ering. The keynote lecture of the Institute was delivered by Dr. John Slaughter, Director of the National Science Foundation, whom we deeply thank for his words of encouragement and commitment to the genetic manipulation of plants.

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.

During the first half century of genetics, coinciding with the first half of this cen tury, geneticists dreamt of the repair of genetic disease by altering or replacing defective genes. H. J. Muller wrote of the great advantages of mutations, "nanoneedles" in his apt term, for delicately probing physiological and chemical processes. In the same spirit, genes could be used to provide treatments of needle point delicacy. Yet, during this period no realistic possibility appeared; it remained but a dream. The situation changed abruptly at the half century. Microbial genetics and its offshoot, cell culture genetics, provided the route. Pneumococcus transformation showed that exogenous DNA could become a permanent part of the genome; yet attempts to reproduce this in animals produced a few tantalizing hints of success, but mostly failures. Transduction, using a virus as mediator, offered a better op portunity. The fITSt reproducible in vivo gene therapy in a whole animal came in 1981. This was in Drosophila, with a transposable element as carrier. Flies were "cured" of a mutant eye color by incorporation of the normal allele, and the effect was transmissible, foreshadowing not only somatic, but germ line gene therapy. At the same time, retroviruses carrying human genes were found to be ex tremely efficient in transferring their contents to the chromosomes of cultured cells.

Critically acclaimed for more than 25 years, the Methods in Cell Biology series provides an indispensable tool for the researcher. Each volume is carefully edited by experts to contain state-of-the-art reviews and step-by-step protocols. Techniques are described completely so that methods are made accessible to users. Describes both well-established and novel recombinant vector systems for expression of proteins Presents methods for efficient delivery of recombinant genes into differentiated cells, tissues, and whole animals Covers high-level and inducible systems, plus assays for protein expression Provides beginning and advanced investigators and students with the information they need to choose the optimal viral or plasmid system for their protein Practical, benchtop-style presentation works in lab and in the classroom