CSH protocols最新文献

INTRODUCTIONDictyostelium discoideum is a unicellular eukaryote often referred to as a "social ameba" because it can form a multicellular structure when nutrient conditions are limiting. General principles for cell-to-cell communication, intracellular signaling, and cytoskeletal organization during cell motility have been derived from cellular and molecular studies of Dictyostelium and have been found to be conserved across all eukaryotes. Dictyostelium also provides an excellent model system for the study of phagocytosis, the molecular basis of various human diseases, and the mechanisms of drug action. The availability of a complete genome database and stocks of wild-type and mutant strains make D. discoideum an accessible and powerful model organism. Most Dictyostelium strains used in the laboratory can be grown either with bacteria or in axenic medium. When grown in the presence of bacteria, cells double approximately every 4 h, whereas axenically grown cells double more slowly, every 8-12 h. The cells can be grown in a standard microbiology incubator or on the laboratory bench, provided the room temperature is consistently ~22°C. This protocol describes a variety of methods for growing and maintaining Dictyostelium.

INTRODUCTIONDictyostelium discoideum is a unicellular eukaryote often referred to as a social ameba because it can form a multicellular structure when nutrient conditions are limiting. General principles for cell-to-cell communication, intracellular signaling, and cytoskeletal organization during cell motility have been derived from cellular and molecular studies of Dictyostelium and have been found to be conserved across all eukaryotes. The availability of a complete genome database and stocks of wild-type and mutant strains make D. discoideum an accessible and powerful model organism. Dictyostelium is amenable to genetic manipulations that require the introduction of DNA into cells, such as gene knockout, overexpression, antisense RNA expression, RNA interference (RNAi)-mediated gene knockdown, and restriction-enzyme-mediated mutagenesis. Extraction of genomic DNA is used to clone gene fragments and for analysis of mutants to determine the site of vector integration. Because Dictyostelium cells contain relatively high levels of carbohydrate and nucleases, commercially available DNA preparation kits are not very successful. The DNA isolated according to the following protocol is suitable for digestion by restriction enzymes, amplification by polymerase chain reaction (PCR), and Southern blotting.

INTRODUCTIONThe two-spotted cricket Gryllus bimaculatus De Geer (Orthoptera: Gryllidae), which is one of the most abundant cricket species, inhabits the tropical and subtropical regions of Asia, Africa, and Europe. G. bimaculatus can be easily bred in the laboratory and has been widely used to study insect physiology and neurobiology. Recently, this species has become established as a model animal for studies on molecular mechanisms of development and regeneration because its mode of development is more typical of arthropods than that of Drosophila melanogaster, and the cricket is probably ancestral for this phylum. Moreover, the cricket is a hemimetabolous insect, in which nymphs possess functional legs with a remarkable capacity for regeneration after damage. Because RNA interference (RNAi) works effectively in this species, the elucidation of mechanisms of development and regeneration has been expedited through loss-of-function analyses of genes. Furthermore, because RNAi-based techniques for analyzing gene functions can be combined with assay systems in other research areas (such as behavioral analyses), G. bimaculatus is expected to become a model organism in various fields of biology. Thus, it may be possible to establish the cricket as a simple model system for exploring more complex organisms such as humans.

INTRODUCTIONDevelopmental gene expression is analyzed predominantly via whole-mount in situ hybridization using digoxigenin-labeled RNA probes. This protocol describes how to perform this procedure in Amphimedon queenslandica, including fixation, hybridization, and sectioning of embryonic, larval, and post-larval juvenile stages.

INTRODUCTIONDictyostelium discoideum is a unicellular eukaryote often referred to as a "social ameba" because it can form a multicellular structure when nutrients are depleted from the immediate environment of the cells. In the laboratory, this is accomplished simply by replacing the growth medium with a buffer solution. For best results, it is important to harvest the cells while they are still in exponential growth (1-4 × 10(6) cells/mL). At high cell density, many of the cells in a culture will have initiated development, thus yielding asynchronous development. Dictyostelium cells can be developed on solid media, either on filter paper or on KK2 plates. If only the early stages of development are important (e.g., to study chemotaxis), cells can be developed in suspension. Under these conditions, cells will only progress through the first 6-8 h of development. Addition of cAMP pulses to the starved suspension culture will allow development to progress up to the 12-h stage, corresponding to the beginning of culmination.

INTRODUCTIONDictyostelium discoideum is a unicellular eukaryote often referred to as a social ameba because it can form a multicellular structure when nutrient conditions are limiting. General principles for cell-to-cell communication, intracellular signaling, and cytoskeletal organization during cell motility have been derived from cellular and molecular studies of Dictyostelium and have been found to be conserved across all eukaryotes. The availability of a complete genome database and stocks of wild-type and mutant strains make D. discoideum an accessible and powerful model organism. Dictyostelium is amenable to genetic manipulations that require the introduction of DNA into cells, such as gene knockout, overexpression, antisense RNA expression, RNA interference (RNAi)-mediated gene knockdown, and restriction-enzyme-mediated mutagenesis. Calcium phosphate precipitation is a commonly used method for DNA-mediated transformation in Dictyostelium. Calcium phosphate precipitation produces high-copy-number transformants and is often used for overexpression experiments in conjunction with the G418 resistance gene, which needs to be present at high levels to produce efficient selection.

INTRODUCTIONDictyostelium discoideum is a unicellular eukaryote often referred to as a social ameba because it can form a multicellular structure when nutrient conditions are limiting. General principles for cell-to-cell communication, intracellular signaling, and cytoskeletal organization during cell motility have been derived from cellular and molecular studies of Dictyostelium and have been found to be conserved across all eukaryotes. The availability of a complete genome database and stocks of wild-type and mutant strains make D. discoideum an accessible and powerful model organism. Dictyostelium is amenable to genetic manipulations that require the introduction of DNA into cells, such as gene knockout, overexpression, antisense RNA expression, RNA interference (RNAi)-mediated gene knockdown, and restriction-enzyme-mediated mutagenesis. The extraction of RNA from Dictyostelium is relatively easy because RNA levels are very high in comparison to DNA levels (i.e., ~40 times higher). Certain commercially available kits, such as Trizol (Invitrogen) and RNeasy (QIAGEN) have been used successfully, although lysis conditions need to be adjusted. RNA samples are stable for several years at -80°C in diethylpyrocarbonate (DEPC)-treated H(2)O. For longer-term storage, the RNA pellet can be stored in 100% ethanol at -80°C. Such samples are suitable for Northern blots, reverse transcriptase-polymerase chain reaction (RT-PCR), and microarray analysis of gene expression.

INTRODUCTIONDictyostelium discoideum is a unicellular eukaryote often referred to as a social ameba because it can form a multicellular structure when nutrient conditions are limiting. General principles for cell-to-cell communication, intracellular signaling, and cytoskeletal organization during cell motility have been derived from cellular and molecular studies of Dictyostelium and have been found to be conserved across all eukaryotes. The availability of a complete genome database and stocks of wild-type and mutant strains make D. discoideum an accessible and powerful model organism. Dictyostelium is amenable to genetic manipulations that require the introduction of DNA into cells, such as gene knockout, overexpression, antisense RNA expression, RNA interference (RNAi)-mediated gene knockdown, and restriction-enzyme-mediated mutagenesis. Two commonly used methods for DNA-mediated transformation in Dictyostelium are calcium phosphate precipitation and electroporation. Transformants can then be selected in liquid media or on bacterial plates. The latter method reduces the chances of contamination because the cells are grown in buffered agar containing live or dead bacteria, rather than in a rich broth. This method also facilitates the isolation of clones from transformations because each transformant produces a single colony on the plate instead of the pools of transformants obtained in liquid culture. For gene ablation experiments, it is important to obtain a minimum of two independent clones with the same phenotype to exclude the possibility that the phenotype is due to a nonspecific mutation.

INTRODUCTIONThe Nucleic Acid Programmable Protein Array (NAPPA) approach for producing protein microarrays uses cell-free extracts to transcribe and translate cDNAs encoding target proteins directly onto glass slides. Following array preparation, interactions with a protein of interest (query protein) are detected either by probing an expressed NAPPA slide with the purified query protein or by coexpressing the query protein on the NAPPA slide at the same time that the target proteins are expressed. This protocol describes the coexpression method, which involves adding the gene for the query protein to the cell-free protein expression mix. The amount of query protein that is transcribed and translated from the corresponding plasmid DNA depends on the amount of plasmid DNA used and the size of the protein of interest, among other factors. If too little query protein is expressed, there may be no detectable binding signal. Excessive amounts of protein expression may generate nonspecific background signals. Because the optimum amount of query plasmid varies with each query protein, it is essential to assess empirically the optimal amount of query protein DNA to add to a coexpression experiment.

INTRODUCTIONCtenophores, or comb jellies, are a group of marine organisms whose unique biological features and phylogenetic placement make them a key taxon for understanding animal evolution. These gelatinous creatures are clearly distinct from cnidarian medusae (i.e., jellyfish). Key features present in the ctenophore body plan include biradial symmetry, an oral-aboral axis delimited by a mouth and an apical sensory organ, two tentacles, eight comb rows composed of interconnected cilia, and thick mesoglea. Other morphological features include definitive muscle cells, a nerve net, basal lamina, a sperm acrosome, and light-producing photocytes. Aspects of their development made them attractive to experimental embryologists as early as the 19th century. Recently, because of their role as an invasive species, studies on their role in ecology and fisheries-related fields have increased. Although the phylogenetic placement of ctenophores with respect to other animals has proven difficult, it is clear that, along with poriferans, placozoans, and cnidarians, ctenophores are one of the earliest diverging extant animal groups. It is important to determine if some of the complex features of ctenophores are examples of convergence or if they were lost in other animal branches. Because ctenophores are amenable to modern technical approaches, they could prove to be a highly useful emerging model.