CSH protocols最新文献

INTRODUCTIONPrimary cultures of granule neurons from the post-natal rat cerebellum provide an excellent model system for molecular and cell biological studies of neuronal development and function. The cerebellar cortex, with its highly organized structure and few neuronal subtypes, offers a well-characterized neural circuitry. Many fundamental insights into the processes of neuronal apoptosis, migration, and differentiation in the mammalian central nervous system have come from investigating granule neurons in vitro. Granule neurons are the most abundant type of neurons in the brain. In addition to the sheer volume of granule neurons, the homogeneity of the population and the fact that they can be transfected with ease render them ideal for elucidating the molecular basis of neuronal development. This protocol for isolating granule neurons from post-natal rats is relatively straightforward and quick, making use of standard enzymatic and mechanical dissociation methods. In a serum-based medium containing an inhibitor of mitosis, cerebellar granule neurons can be maintained with high purity. Axons and dendrites can be clearly distinguished on the basis of morphology and markers. For even greater versatility, this protocol for culturing granule neurons can be combined with knockout or transgenic technologies, or used in cerebellar slice overlay assays.

INTRODUCTIONSponges are one of the earliest branching metazoans. In addition to undergoing complex development and differentiation, they can regenerate via stem cells and can discern self from nonself ("allorecognition"), making them a useful comparative model for a range of metazoan-specific processes. Molecular analyses of these processes have the potential to reveal ancient homologies shared among all living animals and critical genomic innovations that underpin metazoan multicellularity. Amphimedon queenslandica (Porifera, Demospongiae, Haplosclerida, Niphatidae) is the first poriferan representative to have its genome sequenced, assembled, and annotated. Amphimedon exemplifies many sessile and sedentary marine invertebrates (e.g., corals, ascidians, bryozoans): They disperse during a planktonic larval phase, settle in the vicinity of conspecifics, ward off potential competitors (including incompatible genotypes), and ensure that brooded eggs are fertilized by conspecific sperm. Using genomic and expressed sequence tag (EST) resources from Amphimedon, functional genomic approaches can be applied to a wide range of ecological and population genetic processes, including fertilization, dispersal, and colonization dynamics, host-symbiont interactions, and secondary metabolite production. Unlike most other sponges, Amphimedon produce hundreds of asynchronously developing embryos and larvae year-round in distinct, easily accessible brood chambers. Embryogenesis gives rise to larvae with at least a dozen cell types that are segregated into three layers and patterned along the body axis. In this article, we describe some of the methods currently available for studying A. queenslandica, focusing on the analysis of embryos, larvae, and post-larvae.

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. D. discoideum and related organisms, known as the Dictyostelia, have been studied for almost 150 years. The cellular and molecular aspects of their multicellular lifestyle have been studied in detail, and general principles for cell-to-cell communication, intracellular signaling, and cytoskeletal organization during cell motility have been derived from this work and have been found to be conserved across all eukaryotes. The bacteriovore nature of the unicellular stage provides an excellent model in which to study phagocytosis and the mechanisms of bacterial virulence. D. discoideum has also been used successfully to explore the molecular basis of various human diseases, as well as the mechanisms of drug action and the pathways that lead to resistance to certain therapeutic agents. The availability of a complete genome sequence has further widened the scope of studies using D. discoideum. A large potential for secondary metabolism has become apparent, which opens the door to discovering new compounds with potential medical applications. Numerous putative orthologs of genes responsible for diseases in humans, but whose molecular functions are still uncharacterized, are present in the D. discoideum genome. Finally, the availability of community resources, including the genome database dictyBase and the Dicty Stock Center, makes D. discoideum an easily accessible and powerful model organism to study.

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, the identification of protein interactions using NAPPA can be accomplished by either of two general schemas. The first is to probe an expressed NAPPA slide with a purified protein of interest (the query protein) and look for interactors. Signals of these interactions can be detected either by directly labeling the query protein or by using a labeled antibody either to the protein itself or to a tag on the protein. This approach works well when there is access to the purified protein, and it has the advantage that users can test query protein binding with and without post-translational modifications or under a variety of binding conditions. The second schema entails coexpressing the query protein on the NAPPA slide at the same time that all the target proteins are expressed. This article describes the advantages of using a coexpressed query protein and provides advice on choosing a suitable epitope tag.

INTRODUCTIONFertilization occurs internally in Amphimedon and embryos are brooded in multiple chambers throughout the adult. Each chamber contains a mixture of developmental stages, from egg to late ring stages (i.e., prehatch late embryos). At the end of embryogenesis, swimming parenchymella larvae emerge from the adult. After several hours in the water column, the larvae settle and metamorphose into juvenile sponges. This protocol details how to obtain Amphimedon larvae and post-larvae/juveniles as well as embryos. Once isolated, these biological stages can be used for a variety of molecular and cellular analyses.

INTRODUCTIONAlthough attempts to culture prepigmentation-stage embryos (i.e., blastulas and early gastrulas) outside of brood chambers have so far been unsuccessful in Amphimedon, it is possible to manipulate embryos within the brood chamber and follow their development under laboratory conditions. This protocol describes microinjection of lipophilic tracers such as 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) into embryos embedded in their native brood chamber. DiI does not appear to perturb embryonic development and is relatively resistant to photobleaching. As long as care is taken not to damage the fragile embryos during observation and photography, the same embryo can be photographed multiple times, permitting its development to be tracked (up to 4 wk) from early cleavage stages to hatching of free-swimming parenchymella larvae. The embryos or larvae also can be fixed without loss of fluorescence. This method also can be used to deliver other types of solutions to embryos or individual cells of early embryos.

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. Dictyostelium can be grown axenically or in the presence of bacteria, either on agar plates or in suspension. Because Dictyostelium growth rates are relatively slow compared to those of bacteria or yeast, laboratories commonly maintain stocks of growing cultures in order to start experiments rapidly. However, it is important to remember that the genome of Dictyostelium, like that of any living organism, is subject to genetic modification. It is well documented that cell lines that are kept in culture for an extended period of time exhibit undesirable changes that yield unreliable experimental results (Hughes et al. 2007). Dictyostelium strains from different laboratories are known to contain various large genome duplications, presumably due to clone selection. Thus, good handling of the cells is essential. To obtain consistent results, new cultures must be started every 2-4 wk, and cultures should never be allowed to grow beyond 4 × 10(6) cells/mL. If overgrowth occurs, a new culture should be started. This protocol describes two methods for preparing long-term stocks of Dictyostelium, either as frozen cells or as spores.

INTRODUCTIONIn the salt gradient dialysis method, purified core histones are incubated with a DNA template in a buffer containing a high concentration of NaCl. As the salt is slowly dialyzed away, nucleosomes spontaneously assemble on the DNA, and their translational positioning along the DNA is directed by the DNA sequence. In the absence of nucleosome-positioning elements (e.g., 5S rDNA genes), the nucleosomes can adopt a closely packed nonphysiological structure with little space between the nucleosomes. Removal of remaining free histones, as well as templates with closely packed nucleosomes, can be achieved by fractionation over sucrose gradients. The chromatin assembled in these reactions can then be analyzed using micrococcal nuclease digestion. Salt dialysis reconstitutions are easy to perform, but they are time-consuming because of multiple changes of the dialysis buffer. The reconstitution method presented here takes a total of 1.5-2 d to complete.

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. Identification of protein interactions can be accomplished either by probing an expressed NAPPA slide with a purified protein of interest (the 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 detection of query protein following coexpression of the query protein on the NAPPA array.

INTRODUCTIONCore histones can be purified from a variety of cell sources, including Drosophila embryos, HeLa tissue culture cells, calf thymus, or chicken erythrocytes. Chick erythrocytes are an excellent source of cellular histones: Large quantities of source material are readily obtainable, the purified histones have low levels of post-translational modifications, and linker histones can also be purified from the same cell sample. Also, avian histones have an amino acid sequence identical to that of human histones. Histone stocks can be stored successfully for more than a year at 4°C and for several years at -20°C. With this protocol, 200 mL of blood usually yields in excess of 50 mg of purified histone octamers. Additional optional procedures are also presented for the purification of H1 and H5 linker histones, as well as for the preparation of H3/H4 tetramers and H2A/H2B dimers.