实验基础

The development of transfection procedures that optimize the efficiency of recombination events is central to the isolation of recombinant baculoviruses for foreign gene expression. A variety of transfection methods, which were first developed for mammalian cell lines, have be ...

The baculovirus expression system is now a widely accepted tool for the expression of recombinant proteins with many features to recommend it. Selection of recombinants is rapid compared to mammalian expression systems, whereas the capacity to perform many posttranslational modi ...

The classical method for making a recombinant baculovirus relies on homologous recombination in insect cells to replace a segment of the baculovirus genome with the corresponding segment from a transfer vector that has been modified to include a foreign gene. The frequency of this recomb ...

The baculovirus expression system has proven extremely useful in the investigation of the function and structure of a wide variety of proteins produced by the expression of a foreign gene (1). Essential to this system is the isolation of a recombinant baculovirus expressing the foreign gene. ...

The response of Borrelia burgdorferi to the challenge of reactive oxygen species (ROS) is a direct result of its limited biosynthetic capabilities and lack of biologically significant levels of intracellular Fe. In other bacteria, the major target for oxidative damage is DNA as a conseque ...

Methods for genetic manipulation of Staphylococci have advanced considerably over the past decade. Transduction is the most common and simplest method for transferring plasmid DNA or chromosomal markers between strains of Staphylococcus aureus. However, some clinical isola ...

Species of the genus Lactobacillus are used worldwide in the production of fermented food or fodder from raw agricultural materials (1). To obtain fermentation products that are reproducible and of high quality, fermentations are initiated by the addition of well-defined starter cult ...

Yeast shuttle vectors are common tools in molecular and cellular studies of Saccharomyces cerevisiae, including studies of gene structure-function relationships, DNA repair, and recombination (1). Shuttle vectors are manipulated easily in vitro, introduced into Escherich ...

Since first isolated and cultured, there have been numerous reports stating the in vitro susceptibilities of Helicobacter pylori to a wide range of antibacterial agents (1,2), bismuth compounds (3), and compounds inhibiting acid secretion (4).

Baculovirus-based insecticides are currently being used worldwide, and new products are in development in many countries. The most dramatic examples of successful baculovirus insecticides are found in soybean in Brazil and cotton in China. Production of baculoviruses is general ...

Cytokines elicit responses in target cells by inducing changes in gene expression. For interferons (IFNs), this involves receptor-mediated activation of specific transcription factors, which then translocate into the nucleus to bind to cognate gene elements in the promoters of IFN ...

Investigating the cell biology of gene expression requires methodologies for localizing RNA relative to proteins involved in RNA transcription, processing, and export. Adenovirus is an important model system for the analysis of eukaryotic gene expression and is also being used to in ...

RNA interference (RNAi) has attracted a lot of interest during recent years as a method to knock-down gene expression and as a possible antiviral system. Here we present a collection of in vitro methods to study RNAi and the effect of an adenovirus infection on RNAi. We describe methods to measure the two k ...

This chapter describes the techniques used to study nucleolar-localized proteins. The chapter starts with cloning of viral proteins for expression in mammalian cells as fusion proteins to well-characterized tags such as enhanced green fluorescence protein (EGFP). This follows on ...

Co-immunoprecipitation is a common method used to determine protein—protein interactions. This method typically exploits the highly specific interaction between a monoclonal antibody and a protein of interest. Providing that the antibody—protein interaction does not int ...

Adenoviruses (Ads) are small DNA tumor viruses that have played a pivotal role in understanding eukaryotic cell biology and viral oncogenesis. Among other cellular pathways, Ad usurps cell cycle progression following infection. Likewise, progression of the viral infection is infl ...

The encapsidation of adenovirus DNA into virus particles depends on cis-acting sequences located at the left end of the viral genome. Repeated DNA sequences in the packaging domain contribute to viral DNA encapsidation, and several viral proteins bind to these repeats when analyzed using ...

Lipopolysaccharide (LPS) is the signature glycolipid isolated from almost all Gram-negative bacteria. LPSs are well known for their ability to elicit the release of cytokines from eukaryotic cells including macrophages, neutrophils, and epithelial cells. LPS can be isolated free of ...

Streptococcus pyogenes secretes various proteins to the extracellular environment. During infection, these proteins interact with human macromolecules and contribute to pathogenesis. We describe a proteomic approach routinely used in our laboratory to characterize cu ...

The analysis of the expression of virulence genes and the elucidation of metabolic and regulatory pathways of Staphylococcus aureus provide us with important information about the interaction between the pathogen and its host, mechanisms by which this organism causes diseases, and t ...