Gene Transfer and Expression in Mammalian Cells

Front Cover
S.C. Makrides
Newnes, Oct 24, 2003 - Science - 722 pages
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The production of proteins in mammalian cells is an important tool in numerous scientific and commercial areas. For example, proteins for human therapy, vaccination or diagnostic applications are typically produced in mammalian cells. Gene cloning, protein engineering, biochemical and biophysical characterization of proteins also require the use of gene expression in mammalian cells. Other applications in widespread use involve screening of libraries of chemical compounds in drug discovery, and the development of cell-based biosensors.

This book presents a state-of-the-art comprehensive coverage of the technical aspects of gene expression in mammalian cells, written by experienced scientists working at the forefront of the field.
 

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Contents

Expression in primary cell cultures
Expression in hippocampal slice cultures
SFV vectors in vivo
Safety of SFV vectors
SFV vectors in gene therapy
SFV vectors as tools for virus assembly
Introduction 2 Baculoviruses as insect cell expression vectors 3 Molecular biology of virus vector construction
Novel technologies

mRNA and protein stability
Coordinated expression of multiple genes
Herpes simplex virus
Introduction
Herpes Simplex Virusrelevant basic biology
Using HSV1 to make gene therapy vectors
Applications of HSV vectors in the nervous system
Applications outside the nervous system
Virusbased vectors for gene expression in mammalian
Protein production by largescale mammalian cell
Adenoviral vectors
Virusbased vectors for gene expression in mammalian
Gene delivery
Virusbased vectors for gene expression in mammalian
Gene expression
Production and analyses of viral particles
Conclusion
Vaccinia virus
Introduction
Vaccinia virus molecular biology
Construction of recombinant poxvirus vectors
Chimeric VVbacteriophage expression vectors
Improved safety of VV vectors
Nonvaccinia poxvirus vectors
Laboratory and clinical applications
Baculovirus
Baculoviruses as mammalian cell expression vectors
Characteristics of baculovirusmediated mammalian gene delivery
Applications of the baculovirus mammalian gene delivery vector
Points to consider
Assay development
Coronavirus
Introduction
Coronavirus pathogenicity
Molecular biology of coronavirus
Helperdependent expression systems
Single genome coronavirus vectors
Optimization of transcription levels
Modification of coronavirus tropism and virulence
Expression systems based on arteriviruses
Conclusions
Poliovirus
Sindbis virus 1 Introduction
Viral genetic elements
Packaging of replicons
Effects of alphaviruses and replicons on infected cells
Expression in neurons
Insect and crustacea
Vaccines for infectious diseases and cancer
Perspectives
Semliki Forest virus
Introduction
Expression of topologically different proteins
Host cell range
Scaleup of protein production
Conclusions
Retrovirus
Introduction
Biology of retroviruses
Development of recombinant retrovirus vectors
A poliovirus vectorbased HIV vaccine 3 Poliovirus vector development and its immunogenic potential
Expression of the transgene
Targeting
Conclusion and perspectives
Lentiviruses
Introduction
Genetic structure and biology of lentiviruses
HIV1derived vector packaging system
Lentiviral vector production
Pseudotyped vectors
Expression from lentiviral vectors
Gene transfer applications 8 Conclusion
Methods for DNA introduction into mammalian cells
Barriers to successful transfection
Comparison of available methods
Applications
Lipid reagents for DNA transfer into mammalian cells
Introduction
General structure of cationic lipids
Formulation and physicochemical properties
Mechanisms of in vitro transfection
In vivo administration
Targeting
New strategies to improve in vivo transfection
Conclusion
Reporter genes for monitoring gene expression in mammalian cells
Introduction
Reporter gene vectors and fusions
Green fluorescent protein
Luciferases
Alkaline phosphatase
Chloramphenicol acetyltransferase
βGalactosidase
Gene transfer and gene amplification in mammalian cells
Cotransfer of multiple plasmidsviruses as an attractive
Use of scaffoldmatrixattachment regions for protein
Chromatin insulators and position effects
Locus control regions
Pathways and functions of mammalian protein
Metabolic engineering of mammalian cells for higher
Optimization of plasmid backbone for gene expression
Protein production in transgenic animals
Strategies for the purification of recombinant proteins
Product recovery and initial purification
Purification of proteins from culture media
Purification of proteins from cell lysates
Volume reduction and partial purification
Chromatographic purification of recombinant proteins
Product stability and longterm storage
Subject Index

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