实验动物总则

The ability to construct recombinant alleles efficiently in strains of interest, particularly unmarked deletions that reduce the potential for polar effects, is essential to studies of both pathogenesis and basic bacterial physiology. Here we describe a three-phase approach for g ...

Many Gram-positive and Gram-negative bacteria possess natural competence mechanisms for DNA �capture and internalization that play an important role in diversifying adaptation of bacteria through horizontal gene transfer. Natural transformation and other mechanisms of ...

Metabolic pathways and fluxes can be analyzed under in vivo conditions by incorporation experiments using general 13C-labeled precursors. On the basis of the isotopologue compositions in amino acids or other metabolites, the incorporation rates of the supplied precursors and the p ...

This chapter describes methods for culturing Legionella pneumophila in both complex and defined media. The first protocol describes the use of buffered charcoal yeast extract (BCYE) agar, the solid medium that is most commonly used for culturing L. pneumophila. The next procedure detai ...

The production of outer membrane vesicles (OMVs) is a widespread phenomenon employed by bacteria to secrete cell envelope components into the environment. A contribution of Legionella pneumophila OMVs to the pathogenesis of Legionnaires’ disease is likely due to the high number of vir ...

In aquatic environments such as water distribution systems, Legionella pneumophila persistence is �correlated to the presence of a biofilm. The method described here permits the formation of a monospecies L. pneumophila biofilm in microplates, enabling the screening of multiple p ...

Biofilm formation could be studied in various conditions. Most of the studies with Legionella pneumophila used monospecies biofilm in culture media. In some cases, it is important to study bacteria in conditions more close to environmental conditions. In this paper, we describe protoco ...

Legionella pneumophila infects and replicates in environmental protozoa and metazoan macrophages within a specific vacuole. The infection of phagocytes by L. pneumophila can be assessed by an agar plating assay or by fluorescence microscopy. Here, we describe the analysis of Legion ...

RNA interference (RNAi) is the process of specific gene silencing by the use of double-stranded RNA (dsRNA). In cultured Drosophila cells, RNAi methodologies are well established and easily executed: dsRNA, when added to the cell culture medium, is efficiently internalized by the cells and, ...

The lipopolysaccharide(LPS) of Legionella spp. is an immuno-dominant antigen and the basis for Legionella pneumophila serogroup classification. The LPS shows a peculiar structure composed of a very hydrophobic lipid A acylated by long chain fatty acids and an O-antigen-specific ch ...

By means of the Icm/Dot type IV secretion system Legionella pneumophila translocates several effector proteins into host cells, where they anchor to the cytoplasmic face of the LCV membrane by binding to phosphoinositide (PI) lipids. Thus, phosphatidylinositol-4-phosphate anch ...

The study of transcriptome responses can provide insight into the regulatory pathways and genetic factors that contribute to a specific phenotype. For bacterial pathogens, it can identify putative new virulence systems and shed light on the mechanisms underlying the regulation of vi ...

Transcriptional and posttranscriptional regulators play a critical role in allowing a bacterium to adapt to the diverse environments and conditions it encounters. In order to characterize the role of these regulators the identification of their specific interaction partners is ...

Legionella pneumophila the causative agent of Legionnaires’ disease, actively manipulates host cell processes to establish a replication niche inside host cells. The establishment of its replication niche requires a functional Icm/Dot type IV secretion system which transloc ...

Preclinical evaluation of novel anti-smallpox vaccines and antiviral treatments often rely on mouse �challenge models using pathogenic vaccinia virus, such as Western Reserve (WR) strain or other orthopoxviruses. Traditionally, efficacy of treatment is evaluated using var ...

Ectromelia virus infections in the laboratory mouse have emerged as a valuable model to investigate human orthopoxvirus infections to understand the progression of disease, to discover and characterize antiviral treatments, and to study the host–pathogen relationship as it rel ...

Mouse models of immunology are frequently used to study host responses to poxviruses or poxvirus-based recombinant vaccines. In this context, the magnitude of CD8+ T cell responses is often of interest. Methods to evaluate CD8+ T cell responses extend from those that rely on indirect measurem ...

Monoclonal antibodies to specific vaccinia virus (VACV) proteins are valuable reagents in studies of VACV. In this chapter, we describe methods of generating a panel of monoclonal antibodies that recognize a variety of VACV proteins in their native conformation in infected cells. The ant ...

In recent years, there have been numerous unprecedented technological advances in the field of molecular biology; these include DNA sequencing, mass spectrometry of proteins, and microarray analysis of mRNA transcripts. Perhaps, however, it is the area of genomics, which has now genera ...

Antigen presentation to T lymphocytes is the seminal triggering event of the specific immune response, and poxviruses encode immunomodulatory genes that disrupt this process. Discovery of viral proteins that interfere with steps in the antigen presentation process requires a rob ...