CSH protocols最新文献

INTRODUCTIONTransgenic mice possess enormous scientific potential. However, the need to maintain breeding stocks to prevent the loss of unique mutants (particularly if they are not in current use for experiments) is a major challenge. Cryopreservation of spermatozoa or preimplantation embryos is a valuable tool to address this issue, with cryopreservation of embryos in the two- to eight-cell stage being the most common method. Cryopreserved samples can be stored indefinitely in liquid nitrogen, and cryopreserved embryos can be shipped easily. Colonies of a transgenic line need not be maintained if a sufficient stock of embryos has been cryopreserved. After revitalizing preserved samples, embryo transfer must be used to re-establish the line. However, considerable effort is often needed to obtain a sufficient number of embryos. This protocol describes the methods necessary to collect and cryopreserve eight-cell embryos and to handle cryopreserved samples, as well as subsequent procedures needed to revitalize and re-derive transgenic lines.

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. This protocol describes the use of electroporation for DNA-mediated transformation in Dictyostelium.

INTRODUCTIONBone marrow-derived macrophages (BMM) are primary macrophage cells, derived from bone marrow cells in vitro in the presence of growth factors. Macrophage colony-stimulating factor (M-CSF) is a lineage-specific growth factor that is responsible for the proliferation and differentiation of committed myeloid progenitors into cells of the macrophage/monocyte lineage. Mice lacking functional M-CSF are deficient in macrophages and osteoclasts and suffer from osteopetrosis. In this protocol, bone marrow cells are grown in culture dishes in the presence of M-CSF, which is secreted by L929 cells and is used in the form of L929-conditioned medium. Under these conditions, the bone marrow monocyte/macrophage progenitors will proliferate and differentiate into a homogenous population of mature BMMs. The efficiency of the differentiation is assessed using fluorescence-activated cell sorting (FACS) analysis of Mac-1 and 4/80 surface antigen expression. Once differentiated, the BMMs are suitable for numerous types of experimental manipulations, including morphological, gene expression, and physiological studies. For example, phagocytic cells such as macrophages have a unique ability to ingest microbes. We describe a test for the phagocytic efficiency of BMMs by exposing them to fluorescently labeled yeast zymosan bioparticles. Also, a method to deliver DNA or small interfering RNAs (siRNAs) into these hard-to-transfect cells is described. Finally, the proliferation of the BMMs is assayed using carboxyfluorescein succinimidyl ester (CFSE), a fluorescein derivative that partitions equally between daughter cells after cell division.

INTRODUCTIONThe distribution of Amphimedon queenslandica is patchy on coral reefs in the Great Barrier Reef, with small, localized populations detected in shallow, still water reef-flat environments. A. queenslandica is a spermcast spawner, in which fertilization occurs internally. Sperm presumably originate from neighboring reproductive individuals within the population. The ability to genotype individual embryos within a single brood chamber has the potential to shed light on the fertilization biology and generation/maintenance of genetic diversity in this sessile invertebrate. Here, we describe a protocol for rapidly genotyping individuals using polymorphic microsatellite loci. The loci are amplified by PCR using a pair of primers specifically designed for the region of interest with a fluorescent dye attached to the 5'-end to enable easy detection of the amplified product. An advantage of this procedure is that fluorescently labeled PCR products can be combined (i.e., multiplexed) to reduce time and cost when using the genotyping machine. The dye label and size of the product must be taken into consideration when multiplexing. For example, three differently labeled PCR products can be multiplexed, or PCR products with the same label can be multiplexed as long as the allelic size ranges do not overlap. The amount of each cleaned, labeled PCR product added to the multiplex must be optimized depending on the dye and the PCR efficiency.

INTRODUCTIONDue to their large size and long generation times, chondrichthyans have been largely ignored by geneticists. However, their key phylogenetic position makes them ideal subjects to study the molecular bases of the important morphological and physiological innovations that characterize jawed vertebrates. Such analyses are crucial to understanding the origin of the complex genetic mechanisms unraveled in osteichthyans. The small spotted dogfish Scyliorhinus canicula, a representative of the largest order of extant sharks, presents a number of advantages in this context. Due to its relatively small size among sharks, its abundance, and easy maintenance, the dogfish has been an important model in comparative anatomy and physiology for more than a century. Recently, revived interest has occurred with the development of large-scale transcriptomic and genomic resources, together with the establishment of facilities allowing massive egg and embryo production. These new tools open the way to molecular analyses of the elaborate physiological and sensory systems used by sharks. They also make it possible to take advantage of unique characteristics of these species, such as organ zonation, in analyses of cell proliferation and differentiation. Finally, they offer important perspectives to evolutionary developmental biology that will provide a better understanding of the origin and diversifications of jawed vertebrates. The dogfish whole-genome sequence, which may shortly become accessible, should establish this species as an essential shark reference, complementary to other chondrichthyan models. These analyses are likely to reveal an organism of an underestimated complexity, far from the primitive prototypical gnathostome anticipated in gradistic views.

INTRODUCTIONIn ovo RNA interference (RNAi) is a method for silencing a gene of interest using a combination of in ovo injection and electroporation in avian embryos. Here we describe gene silencing in the developing spinal cord, but the procedure can easily be adapted to other parts of the nervous system. Double-stranded RNA (dsRNA) derived from the gene of interest is injected into the developing spinal cord of the chicken embryo, and is followed by electroporation to allow for the uptake of the dsRNA. With this method, temporal as well as spatial control of gene silencing is possible. The time point of injection should be chosen according to the expression profile of the gene or the half-life of the protein. Proteins with slow turnover may require RNAi at earlier stages, ideally before the onset of gene expression. The electroporation parameters can be adjusted such that only a specific population of neurons is targeted in the spinal cord.