实验基础

Respiratory chain dysfunction is an important contributor to human pathology (1–3). The generation of animal models has much facilitated in-depth studies of pathogenetic mechanisms in mitochondrial disease (4). The function of the respiratory chain is subject to the dual genetic con ...

Mitochondrial DNA (mtDNA) nucleoids have been isolated from several organisms, including rat (liver) (1), Physarum polycephalum (2), Saccharomy- ces cerevisiae (3) and Pichia jadinii (4). Most methods for nucleoid isolation have utilized detergent extraction and sucrose gradie ...

The development of baculovirus expression vector systems has accompanied a rapid expansion of our knowledge about the genes, their function, and regulation in insect cells. Classification of these viruses has also been refined as we learn more about differences in gene content between i ...

The insect cell culture/baculovirus system has three primary applications: (1) recombinant protein synthesis, (2) biopesticide synthesis, and (3) as a model system (e.g., for studying apoptosis). The fundamental techniques involved in these applications are described througho ...

The production of a recombinant baculovirus expression vector normally involves mixing infectious virus DNA with a plasmid-based transfer vector and then cotransfecting insect cells to initiate virus infection. The aim of this chapter is to provide an update on the range of baculovirus ...

Although there are several different methods available of making recombinant baculovirus expression vectors (reviewed in Chapter 3), all require a stage in which insect cells are transfected with either the virus genome alone (Bac-to-Bac� or BaculoDirect™, Invitrogen) or virus geno ...

This chapter lists the known cell lines from Lepidoptera, largely based on previous compilations of insect cell lines published by W. Fred Hink. The official designation is given for each cell line as well as the species, tissue source, and, when known, the susceptibles to baculoviruses.

BacMams are modified baculoviruses that contain mammalian expression cassettes for gene delivery and expression in mammalian cells. The BacMam system combines the advantages of viral transient expression, ease in generation, and a wide cell tropism. It enables rapid, facile, and fle ...

Lepidopteran insect cell culture technology has progressed to the point of becoming an essential part of one of the most successful eukaryotic expression systems and is increasingly used industrially on a large scale. Therefore, there is a constant need for convenient and low-cost cultu ...

This chapter describes procedures for initiating new cell lines from lepidopteran larval tissues. The internal morphology is described along with methods for treating excised tissues and the primary cultures. Advice on culture medium and the tissues that will provide the best chance f ...

The various methods for maintaining (a.k.a., subculturing, splitting, or passaging) established lepidopteran cell lines are described. Three procedures are presented that are appropriate for different cell lines dependent upon the growth characteristics (in particular, ce ...

The production of recombinant proteins using the baculovirus expression vector system in large-scale agitated bioreactors is discussed in this chapter. Detailed methods of the key stages of a batch process, including host cell growth, virus stock amplification and quantificatio ...

Improved methods of baculovirus cloning and insect cell culture and their commercialization have made the use of the baculovirus expression vector system (BEVS) a routine tool for the production of preparative quantities of recombinant protein. This chapter outlines basic techni ...

Wave Bioreactors are relatively new to the field of protein production from insect cells infected with recombinant baculoviruses. Various sizes are available to support expression needs from small to large scale. They offer many advantages over stirred tank and airlift reactors, inc ...

With an increasing need for functional analysis of proteins, there is a growing demand for fast and cost-effective production of biologically active eukaryotic proteins. The baculovirus expression vector system is widely used, and in the vast majority of cases cultured insect cells have ...

The ability to make a large variety of virus-like particles (VLPs) has been successfully achieved in the baculovirus expression vector system (BEVS)/insect cell system. The production and scale-up of these particles, which are mostly sought as candidate vaccines, are currently being ad ...

Techniques and experimental applications are described for exogenous protein expression in Drosophila cell lines. Ways in which the Drosophila cell lines and the baculovirus expression vector system differ in their applications are emphasized.

The baculovirus expression vector system is widely used to produce large quantities of recombinant proteins. However, yields of extracellular and membrane-bound proteins obtained with this system often are very low, possibly because of the adverse effects of baculovirus infecti ...

Baculoviruses are widely used both as protein expression vectors and as insect pest control agents. This section provides an overview of the baculovirus lifecycle and use of baculoviruses as insecticidal agents. This chapter includes discussion of the pros and cons for use of baculoviru ...

The lepidopteran insect cells used with the baculovirus expression vector system (BEVS) are capable of synthesizing and accurately processing foreign proteins. However, proteins expressed in baculovirus-infected cells often fail to be completely processed, or are not proces ...