The decision to write a book about the practical aspects of the baculovirus expression system stems from the numerous phone calls for help we have had, and from the many visitors to our labora tories requiring assistance to find the elusive polyhedrin-negative virus containing their favourite gene. We have also organized two expression system workshops and from the manuals we wrote for these, it seemed a logical progression to extend them into book form. We appreciate that those who are 'old-hands' at the baculovirus expression system may have differing views on some of our procedures, but the…mehr
The decision to write a book about the practical aspects of the baculovirus expression system stems from the numerous phone calls for help we have had, and from the many visitors to our labora tories requiring assistance to find the elusive polyhedrin-negative virus containing their favourite gene. We have also organized two expression system workshops and from the manuals we wrote for these, it seemed a logical progression to extend them into book form. We appreciate that those who are 'old-hands' at the baculovirus expression system may have differing views on some of our procedures, but the methods in this book are presented in the light of our own experiences in the laboratory and from our practical workshops, and we hope that the book will be especially useful to those new to the system. The first three chapters give the background information to the baculovirus expression system, and includes advice on how to choose the right transfer vector and discusses the various methods that are available to select recombinant viruses. The practical chapters concentrate on those aspects which are novel to the baculovirus system (insect cell culture, virus amplification and titration, etc. ) and, in general, leave the standard molecular biological techniques to the other excellent laboratory manuals that are available. However, for completeness sake and to avoid constant reference to other manuals, we have included brief details of some standard techniques where they are integral to the success of the baculovirus protocols.
1 The baculoviruses.- 1.1 Introduction.- 1.2 Isolation and host range.- 1.3 Structure and classification.- 1.4 Baculovirus replication in vivo.- 1.5 Baculovirus replication in vitro.- 1.6 Genetic engineering of baculovirus insecticides.- 2 The development of baculovirus expression vectors.- 2.1 Introduction and historical perspective.- 2.2 The merits of the baculovirus expression system.- 2.3 General principles for inserting foreign genes into the baculovirus genome.- 2.4 Baculovirus transfer vectors.- 2.5 Selection of recombinant viruses.- 3 Processing of foreign proteins synthesized using baculovirus vectors in insect cells.- 3.1 Introduction.- 3.2 Glycosylation.- 3.3 Phosphorylation, acylation and amidation.- 3.4 Proteolytic processing.- 3.5 Cellular targeting and secretion.- 3.6 Tertiary and quaternary structure formation.- 3.7 Expression of viral genes.- 3.8 Expression of bacterial and fungal genes.- 3.9 Post-transcriptional processing.- 4 Construction of transfer vectors containing the foreign gene.- 4.1 Introduction.- 4.2 Isolation of foreign gene coding sequences.- 4.3 Modifying the ends of DNA molecules.- 4.4 Preparation of the transfer vector.- 4.5 DNA ligations.- 4.6 Transformation of bacteria.- 4.7 Screening for recombinant baculovirus transfer vectors.- 4.8 Analysis of recombinant transfer vectors.- 4.9 Isolation of highly purified plasmid DNA (maxi-preps).- 5 Insect cell culture media and maintenance of insect cell lines.- 5.1 Introduction.- 5.2 Cell lines.- 5.3 Culture media.- 5.4 Preparation of culture media.- 5.5 Glassware and disposable plasticware.- 5.6 Insect cell culture.- 5.7 A guide to Sf cell seeding densities for experimental work.- 5.8 Freezing, storage and recovery of insect cells in liquid nitrogen.- 5.9 A guide to adapting cells to serum-free media.- 6 Propagation, titration and purification of AcMNPV in cell culture.- 6.1 Introduction.- 6.2 Infection of cells with virus for experimental work.- 6.3 Titration of virus by plaque-assay.- 6.4 Plaque-picking and plaque-purification.- 6.5 Amplification of virus stocks.- 6.6 Large-scale production of virus for the purification of virus particles.- 6.7 Purification of infectious virus DNA.- 6.8 Titration of virus by TCID50.- 7 Production and selection of recombinant virus.- 7.1 Introduction.- 7.2 Preparation of linear AcMNPV.lacZ (or AcMNPV.SC) DNA.- 7.3 Co-transfection of insect cells.- 7.4 Separation of parental and recombinant viruses by plaque-assay.- 7.5 Plaque-purification and amplification of recombinant virus stocks.- 7.6 Amplification and detection of recombinant viruses by limiting dilution and dot-blot hybridization.- 8 Characterization of recombinant viruses.- 8.1 Introduction.- 8.2 Analysis of recombinant virus genomes.- 8.3 Analysis of foreign gene expression by polyacrylamide gel electophoresis, using unlabelled or radiolabelled cell proteins.- 8.4 Analysis of recombinant protein synthesis in insect cells using immunological techniques.- 8.5 Analysis of post-translational processing events in insect cells.- 8.6 Analysis of transcription in recombinant virus-infected cells.- 9 Scaling up the production of recombinant protein in insect cells; laboratory bench level.- 9.1 Introduction.- 9.2 Large-scale culture of insect cells.- 9.3 The importance of highly infectious virus stocks.- 9.4 Multiplicity of infection.- 9.5 The optimum time to harvest virus-infected cells.- 9.6 Purification of recombinant protein from infected cell cultures.- 10 Propagation of baculoviruses in insect larvae.- 10.1 Introduction.- 10.2 Rearing insects in the laboratory.- 10.3 Infection of insect larvae with polyhedra from cell culture.- 10.4 Purification of polyhedra from infected larvae.- 10.5 Bioassays of polyhedra.- 10.6 Purification of virus particles and DNA from polyhedra.- 10.7 Isolation of virus particles from infected larvae to establish infections in cell culture.- 10.8 Preparation of semi-synthetic insect diet.- 11 Trouble-shooting guide.- 11.1 Introduction.- 11.2 Insertion of foreign gene coding sequences into transfer vectors.- 11.3 Cell culture.- 11.4 Preparation of virus stocks and infectious DNA.- 11.5 Co-transfections.- 11.6 Baculovirus plaque-assays.- 11.7 Screening for recombinant viruses.- 11.8 Instability of recombinant viruses.- 11.9 Poor yields of recombinant protein.- Appendix A list of selected suppliers.- References.
1 The baculoviruses.- 1.1 Introduction.- 1.2 Isolation and host range.- 1.3 Structure and classification.- 1.4 Baculovirus replication in vivo.- 1.5 Baculovirus replication in vitro.- 1.6 Genetic engineering of baculovirus insecticides.- 2 The development of baculovirus expression vectors.- 2.1 Introduction and historical perspective.- 2.2 The merits of the baculovirus expression system.- 2.3 General principles for inserting foreign genes into the baculovirus genome.- 2.4 Baculovirus transfer vectors.- 2.5 Selection of recombinant viruses.- 3 Processing of foreign proteins synthesized using baculovirus vectors in insect cells.- 3.1 Introduction.- 3.2 Glycosylation.- 3.3 Phosphorylation, acylation and amidation.- 3.4 Proteolytic processing.- 3.5 Cellular targeting and secretion.- 3.6 Tertiary and quaternary structure formation.- 3.7 Expression of viral genes.- 3.8 Expression of bacterial and fungal genes.- 3.9 Post-transcriptional processing.- 4 Construction of transfer vectors containing the foreign gene.- 4.1 Introduction.- 4.2 Isolation of foreign gene coding sequences.- 4.3 Modifying the ends of DNA molecules.- 4.4 Preparation of the transfer vector.- 4.5 DNA ligations.- 4.6 Transformation of bacteria.- 4.7 Screening for recombinant baculovirus transfer vectors.- 4.8 Analysis of recombinant transfer vectors.- 4.9 Isolation of highly purified plasmid DNA (maxi-preps).- 5 Insect cell culture media and maintenance of insect cell lines.- 5.1 Introduction.- 5.2 Cell lines.- 5.3 Culture media.- 5.4 Preparation of culture media.- 5.5 Glassware and disposable plasticware.- 5.6 Insect cell culture.- 5.7 A guide to Sf cell seeding densities for experimental work.- 5.8 Freezing, storage and recovery of insect cells in liquid nitrogen.- 5.9 A guide to adapting cells to serum-free media.- 6 Propagation, titration and purification of AcMNPV in cell culture.- 6.1 Introduction.- 6.2 Infection of cells with virus for experimental work.- 6.3 Titration of virus by plaque-assay.- 6.4 Plaque-picking and plaque-purification.- 6.5 Amplification of virus stocks.- 6.6 Large-scale production of virus for the purification of virus particles.- 6.7 Purification of infectious virus DNA.- 6.8 Titration of virus by TCID50.- 7 Production and selection of recombinant virus.- 7.1 Introduction.- 7.2 Preparation of linear AcMNPV.lacZ (or AcMNPV.SC) DNA.- 7.3 Co-transfection of insect cells.- 7.4 Separation of parental and recombinant viruses by plaque-assay.- 7.5 Plaque-purification and amplification of recombinant virus stocks.- 7.6 Amplification and detection of recombinant viruses by limiting dilution and dot-blot hybridization.- 8 Characterization of recombinant viruses.- 8.1 Introduction.- 8.2 Analysis of recombinant virus genomes.- 8.3 Analysis of foreign gene expression by polyacrylamide gel electophoresis, using unlabelled or radiolabelled cell proteins.- 8.4 Analysis of recombinant protein synthesis in insect cells using immunological techniques.- 8.5 Analysis of post-translational processing events in insect cells.- 8.6 Analysis of transcription in recombinant virus-infected cells.- 9 Scaling up the production of recombinant protein in insect cells; laboratory bench level.- 9.1 Introduction.- 9.2 Large-scale culture of insect cells.- 9.3 The importance of highly infectious virus stocks.- 9.4 Multiplicity of infection.- 9.5 The optimum time to harvest virus-infected cells.- 9.6 Purification of recombinant protein from infected cell cultures.- 10 Propagation of baculoviruses in insect larvae.- 10.1 Introduction.- 10.2 Rearing insects in the laboratory.- 10.3 Infection of insect larvae with polyhedra from cell culture.- 10.4 Purification of polyhedra from infected larvae.- 10.5 Bioassays of polyhedra.- 10.6 Purification of virus particles and DNA from polyhedra.- 10.7 Isolation of virus particles from infected larvae to establish infections in cell culture.- 10.8 Preparation of semi-synthetic insect diet.- 11 Trouble-shooting guide.- 11.1 Introduction.- 11.2 Insertion of foreign gene coding sequences into transfer vectors.- 11.3 Cell culture.- 11.4 Preparation of virus stocks and infectious DNA.- 11.5 Co-transfections.- 11.6 Baculovirus plaque-assays.- 11.7 Screening for recombinant viruses.- 11.8 Instability of recombinant viruses.- 11.9 Poor yields of recombinant protein.- Appendix A list of selected suppliers.- References.
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