Recombinant Gene Expression Protocols

The current explosive progress in molecular biological research can be definitively traced to the development of molecular cloning technology. The ability to insert specific gene sequences into cloning vectors and their subse­ quent expansion is the cornerstone of modem molecular biology. A direct practical outcome of molecular cloning technology is its application to ex­ press specific recombinant genes. Currently, recombinant gene products are used in a wide spectrum of applications, including gene therapy, production of bioactive pharmaceuticals, synthesis of novel biopolymers, in agriculture and animal husbandry, and so on. A fundamental requirement for successful recombinant gene expression is the design of the cloning vector and the choice of the host organism for expression. Recombinant Gene Expression Protocols grows out of the need for a laboratory manual that provides the reader the background and rationale, as well as the practical protocols for the preparation of "expression constructs" and their introduction into appropriate host cells and/or organisms. The chap­ ters in this book are grouped by their expression hosts, including E. coli, yeast, mammalian cells, nonmammalian eukaryotes such as plants, Xenopus, and insects, as well as in transgenic organisms. In-depth information is presented on the important characteristics of expression cloning vectors and the various methods for efficiently introducing expression constructs into target cells and/ or organisms. Throughout Recombinant Gene Expression Protocols, the authors have consistently striven for a balanced presentation of both background informa­ tion and actual laboratory details.

Part I. Introduction. Recombinant Gene Expression Protocols: Overview of Experimental Strategies for the Expression of Recombinant Genes, Rocky S. Tuan. Part II. Expression in E. coli. Designing Expression Plasmid Vectors in E. coli, Paulina Balbás. Design of Bacterial Hosts for lac-Based Expression Vectors, Herbert P. Schweizer and RoxAnn R. Karkhoff-Schweizer. Bacteriophage l-Based Expression Vectors, Alan C. Christensen. Strategies for Gene Fusions, Stefan Ståhl, Per-Åke Nygren, and Mathias Uhlén. Application of the E. coli trp Promoter, Daniel G. Yansura and Steven H. Bass. Expression of Proteins in E. coli Utilizing a Dual Promoter-Based Vector: pLACT7, George A. Garcia and Shaorong Chong. Expression and Secretion of Proteins in E. coli, Ophry Pines and Masayori Inouye. Synthetic Two-Cistron Expression System, Brigitte E. Schoner. Expression of cDNA-Encoded Proteins by Cell-Free Transcription and Translation, Christopher Coleclough. Part III. Expression in Yeast. Yeast Plasmids, Keshav K. Singh and Jack A. Heinemann. Inducible Expression Cassettes in Yeast: GAL4, Lawrence M. Mylin and James E. Hopper. Inducible Expression Cassettes in Yeast: ADH2, Virginia L. Price. Constitutive Expression Vectors: PGK, Ian R. Graham and Alistair Chambers. Part IV. Viral Expression Systems. Design and Construction of Recombinant Vaccinia Viruses, Christopher C. Broder and Patricia L. Earl. A Simplified Method of Screening for Isolation of Recombinant Vaccinia Virus, Haifeng Chen and R. Padmanabhan. Design of Retroviral Expression Vectors, Kenine E. Comstock, Nathaniel F. Watson, and John C. Olsen. Use of Defective Herpes-Derived Plasmid Vectors, Filip Lim, Dean Hartley, Philip Starr, Song Song, Phung Lang, Linda Yu, Yaming Wang, and Alfred I. Geller. Part V. Expression in Nonmammalian Higher Eukaryotes. Use of Baculovirus Expression Vectors, David R. O'Reilly. Expression of Exogenous Genes in Xenopus Oocytes, Eggs, and Embryos, Koichiro Shiokawa, Chie Koga, Yuzuru Ito, andMikihito Shibata. Analysis of Heterologous Gene Expression in Xenopus Blastomeres, Sally A. Moody. Part VI. Expression in Mammalian Cells. Overview of Vector Design for Mammalian Gene Expression, Randal J. Kaufman. Experimental Strategies in Efficient Transfection of Mammalian Cells: Calcium Phosphate and DEAE-Dextran, Gregory S. Pari and Wayne A. Keown. Experimental Strategies in Efficient Transfection of Mammalian Cells: Electroporation, Donald C. Chang. Highly Effective Delivery of Foreign DNA to Adherent Cells via Polybrene/DMSO-Assisted Gene Transfer, Rémy A. Aubin, Michael Weinfeld, Marzieh Taghavi, Razmik Mirzayans, and Malcolm C. Paterson. Selection of Transfected Cells: Magnetic Affinity Cell Sorting, Raji Padmanabhan, Snorri S. Thorgeirsson, and R. Padmanabhan. Selection of Transfected Cells and Coamplification of Transfected Genes, Susan E. Kane. Optimization of Growth, Viability, and Specific Productivity for Expression of Recombinant Proteins in Mammalian Cells, Jennie P. Mather, Alison Moore, and Robert Shawley. Part VII. Expression in Transgenic Organisms. Promoters to Express Cloned Genes Uniformly in Drosophila, Jean-Paul Vincent and Charles Girdham. Application of Micromechanical Piercing Structures for Genetic Transformation of Nematodes, Sarwar Hashmi and Randy Gaugler. Strategies in Generating Transgenic Mammals, Olena Jacenko. Use of Gonadal Primordial Germ Cells (PGCs) as Tools for Gene Transfer in Chickens, Nathalie Allioli, Gérard Verdier, and Catherine Legras. Strategies for the Production of Transgenic Chickens, Robert J. Etches and Ann M. Verrinder Gibbins. Part VIII. Expression in Plants. Plant Cell Transfection by Electroporation, William B. Terzaghi and Anthony R. Cashmore. Transformation and Foreign Gene Expression in Plants Mediated by Microprojectile Bombardment, Henry Daniell. Transformation of the Cereals Using Agrobacterium, Jean Gould. Transformation of Microalgae Using Silicon Carbide Whiskers, Terri G. Dunahay, Sally A.