Recombinant Gene Expression Protocols

1997
ISBN: 978-0-89603-480-8
Verlag: Humana Press

Buch, Englisch, Band 62, 521 Seiten, Format (B × H): 160 mm x 241 mm, Gewicht: 1074 g

Reihe: Methods in Molecular Biology

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.

Zielgruppe

Research

Autoren/Hrsg.

Tuan, Rocky S.

Fachgebiete

• Naturwissenschaften Biowissenschaften Zellbiologie
• Naturwissenschaften Biowissenschaften Biochemische Immunologie
• Naturwissenschaften Biowissenschaften Molekularbiologie
• Naturwissenschaften Biowissenschaften Biowissenschaften DNA und Transgene Organismen
• Naturwissenschaften Biowissenschaften Proteinforschung

Inhaltsverzeichnis

Recombinant Gene Expression Protocols.- Expression in E. Coli.- Designing Expression Plasmid Vectors in E. coli.- Design of Bacterial Hosts for lac-Based Expression Vectors.- Bacteriophage ?-Based Expression Vectors.- Strategies for Gene Fusions.- Application of the E. coli trp Promoter.- Expression of Proteins in E. coli Utilizing a Dual Promoter-Based Vector: pLACT7.- Expression and Secretion of Proteins in E. coli.- Synthetic Two-Cistron Expression System.- Expression of cDNA-Encoded Proteins by Cell-Free Transcription and Translation.- Expression in Yeast.- Yeast Plasmids.- Inducible Expression Cassettes in Yeast: GAL4.- Inducible Expression Cassettes in Yeast: ADH2.- Constitutive Expression Vectors: PGK.- Viral Expression Systems.- Design and Construction of Recombinant Vaccinia Viruses.- A Simplified Method of Screening for Isolation of Recombinant Vaccinia Virus.- Design of Retroviral Expression Vectors.- Use of Defective Herpes-Derived Plasmid Vectors.- Expression in Nonmammalian Higher Eukaryotes.- Use of Baculovirus Expression Vectors.- Expression of Exogenous Genes in Xenopus Oocytes, Eggs, and Embryos.- Analysis of Heterologous Gene Expression in Xenopus Blastomeres.- Expression in Mammalian Cells.- Overview of Vector Design for Mammalian Gene Expression.- Experimental Strategies in Efficient Transfection of Mammalian Cells.- Experimental Strategies in Efficient Transfection of Mammalian Cells.- Highly Effective Delivery of Foreign DNA to Adherent Cells via Polybrene/DMSO-Assisted Gene Transfer.- Selection of Transfected Cells.- Selection of Transfected Cells and Coamplification of Transfected Genes.- Optimization of Growth, Viability, and Specific Productivity for Expression of Recombinant Proteins in Mammalian Cells.- Expression in Transgenic Organisms.-Promoters to Express Cloned Genes Uniformly in Drosophila.- Application of Micromechanical Piercing Structures for Genetic Transformation of Nematodes.- Strategies in Generating Transgenic Mammals.- Use of Gonadal Primordial Germ Cells (PGCs) as Tools for Gene Transfer in Chickens.- Strategies for the Production of Transgenic Chickens.- Expression in Plants.- Plant Cell Transfection by Electroporation.- Transformation and Foreign Gene Expression in Plants Mediated by Microprojectile Bombardment.- Transformation of the Cereals Using Agrobacterium.- Transformation of Microalgae Using Silicon Carbide Whiskers.