CSH protocols最新文献

INTRODUCTIONThis protocol describes general strategies for propagating Antirrhinum (snapdragon) species: self- and cross-pollination, cuttings, and grafting. Antirrhinum majus cultivars and some wild species are self-fertile, but they require self-pollination for high seed yields. Although self-fertile, A. majus shows unilateral incompatibility and can only be crossed to other self-incompatible species as the female parent. All Antirrhinum species can be propagated clonally from cuttings. Antirrhinum also readily forms grafts within and between species.

INTRODUCTIONThe metabolic activities of bacteria growing in biofilms result in spatial gradients of oxygen, nutrients, and waste products. Because bacteria respond to local environmental conditions through changes in gene expression, mRNA levels of individual genes may vary spatially among bacteria within the biofilm. This article describes an approach to isolate RNA for quantification from cells at localized sites within biofilms. Biofilm thin sections are generated by embedding biofilms in cryoembedding resin, freezing the embedded biofilms on dry ice, and cutting with a cryomicrotome. The sections are placed on membrane-coated microscope slides and maintained on dry ice. Laser capture microdissection microscopy (LCMM) is used to dissect small subsets of cells at different regions within the biofilms, and RNA is extracted from the samples using either hot phenol or TRI reagent. A TRI reagent-based DNA extraction method is also presented.

INTRODUCTIONMonodelphis domestica, the gray, short-tailed, or laboratory opossum, is the most commonly used laboratory marsupial. In addition to the factors that make it a convenient laboratory animal (small size, ease of care, nonseasonal breeding), it is the first marsupial whose genome has been sequenced. Monodelphis has proven useful as a model organism for studies on spinal cord regeneration, ultraviolet (UV)-induced melanoma, and genetic influences on cholesterol, as well as comparative studies of the immune system. In addition, Monodelphis has been used to understand the basic functions of the olfactory system and the role of various olfactory chemicals in social and reproductive behavior. Recently, Monodelphis has been used to understand fundamental aspects of marsupial development, anatomy, evolution, and evolutionary consequences of the derived marsupial mode of development and reproduction. This protocol details whole-mount in situ hybridization of Monodelphis embryos, but it is broadly applicable to any marsupial. Special conditions have been included throughout the protocol for various stages of marsupial embryos. Nevertheless, whole, preterm embryonic stages (~stage 33 to birth) have proven to be difficult to work with because formation of the cuticle prevents probe and antibody penetration.

INTRODUCTIONThe spider Cupiennius salei, commonly known as the American Wandering Spider, is a particularly useful laboratory model for embryological studies because of the availability of tools to study and manipulate its embryonic development. Cupiennius is used to study axis formation, segmentation, appendage development, neurogenesis, and silk production. These studies contribute to our understanding of the evolution of these processes, but they also help us to understand the origin and diversification of evolutionary novelties. Comparisons between spiders and insects can show the degree of conservation and divergence of developmental mechanisms during arthropod evolution. Any embryological feature conserved between spiders and insects is likely to represent an ancestral feature for arthropods. Comparative molecular embryological work in insects and spiders should eventually allow us to define a molecular archetype for the phylum Arthropoda. This in itself will be a necessary cornerstone for comparing the different metazoan phyla, including chordates. A feature of apoptosis (i.e., cell death) is the cleavage or fragmentation of DNA that occurs in dead or dying cells. This protocol describes the detection of fragmented DNA in whole-mount Cupiennius embryos. The 3'-OH ends of these DNA fragments can be labeled with the terminal deoxynucleotidyl-transferase-mediated dUTP-digoxigenin nick-end labeling (TUNEL) technique. This protocol uses a terminal deoxynucleotidyl transferase to add labeled dUTP to the fragmented DNA, and this label is then detected by immunocytochemistry. The TUNEL technique is a relatively easy way to obtain a reliable picture of the cell death pattern during normal and abnormal development.

INTRODUCTIONThe spider Cupiennius salei, commonly known as the American wandering spider, is particularly useful for embryological studies because of the availability of tools to study and manipulate its embryonic development. Cupiennius is used to study axis formation, segmentation, appendage development, neurogenesis, and silk production. These studies contribute to our understanding of the evolution of these processes, but they also help us to understand the origin and diversification of evolutionary novelties. Comparisons between spiders and insects can show the degree of conservation and divergence of developmental mechanisms during arthropod evolution. Any embryological feature conserved between spiders and insects is likely to represent an ancestral feature for arthropods. Comparative molecular embryological work in insects and spiders should eventually allow us to define a molecular archetype for the phylum Arthropoda. This will be a necessary cornerstone for comparing the different metazoan phyla, including chordates. This protocol describes the detection of proliferating cells in whole-mount Cupiennius embryos. When labeled nucleotides are introduced into mitotically dividing cells, these cells incorporate the labels into the newly synthesized DNA. Thus, only cells that have synthesized DNA after the addition of the label will be detected. This protocol uses 5-bromo-2'-deoxy-uridine (BrdU) as a label that is subsequently detected by immunocytochemistry. BrdU labeling is a relatively easy way to detect cells that have recently synthesized DNA. The main advantage of this technique is that the label accumulates over time and, by varying the incubation time before fixation, an increasingly cumulative picture of cell proliferation activity can be obtained.

INTRODUCTIONTo provide sufficient material for experimentation, a laboratory needs to expand and maintain a colony of planarians. It is crucial to keep a stable, healthy population of animals in a consistent environment to avoid inter-animal variability and modifier effects that can mask true phenotypes from experimental perturbation. In this protocol, we describe basic procedures for establishing and maintaining healthy colonies of Dugesia japonica, Schmidtea mediterranea, and Girardia tigrina (commonly found in the wild and commercially available in the United States). Although the recommendations are based on our optimization of conditions for G. tigrina, many of the procedures (such as food preparation and feeding strategy) can be applied to other species. For best results, the culture water must be carefully monitored and adjusted for each species.

INTRODUCTIONThe spider Cupiennius salei is a useful laboratory model for embryological and physiological studies. Its highly developed sensory organs also make it an excellent model for behavioral studies. Furthermore, Cupiennius has contributed greatly to the study of evolutionary developmental questions. This chelicerate arthropod is particularly useful for such studies because of its phylogenetic position and the availability of tools to study and manipulate its embryonic development.

INTRODUCTIONMonodelphis domestica, the gray, short-tailed, or laboratory opossum, is the most commonly used laboratory marsupial. In addition to the factors that make it a convenient laboratory animal (small size, ease of care, nonseasonal breeding), it is the first marsupial whose genome has been sequenced. Monodelphis has proven useful as a model organism for studies on spinal cord regeneration, ultraviolet (UV)-induced melanoma, and genetic influences on cholesterol, as well as comparative studies of the immune system. In addition, Monodelphis has been used to understand the basic functions of the olfactory system and the role of various olfactory chemicals in social and reproductive behavior. Recently, Monodelphis has been used to understand fundamental aspects of marsupial development, anatomy, evolution, and evolutionary consequences of the derived marsupial mode of development and reproduction. The embryos of Monodelphis, like those of other marsupials, can be cultured in vitro. The length of embryo viability depends in part on the stage at which culture begins, but embryos of different species of marsupials have been cultured for 18 h to almost 72 h. Good culture results for Monodelphis have been obtained using the method presented here. Embryos can be manipulated and then placed in the incubator. We have applied this technique most commonly to embryos at stages 23-25; they have retained viability and normal development through stage 26 when embryos would begin to implant in vivo.

INTRODUCTIONThe spider Cupiennius salei, commonly known as the American wandering spider, is a particularly useful laboratory model for embryological studies because of the availability of tools to study and manipulate its embryonic development. Cupiennius is used to study axis formation, segmentation, appendage development, neurogenesis, and silk production. These studies contribute to our understanding of the evolution of these processes, but they also help us to understand the origin and diversification of evolutionary novelties. Comparisons between spiders and insects can show the degree of conservation and divergence of developmental mechanisms during arthropod evolution. Any embryological feature conserved between spiders and insects is likely to represent an ancestral feature for arthropods. Comparative molecular embryological work in insects and spiders should eventually allow us to define a molecular archetype for the phylum Arthropoda. This in itself will be a necessary cornerstone for comparing the different metazoan phyla, including chordates. This protocol describes the collection and fixation of embryos from C. salei. The fixed embryos can be stored at -20°C for prolonged periods and used for in situ hybridization, in studies of apoptosis using terminal deoxynucleotidyl-transferase-mediated dUTP-digoxigenin nick-end labeling (TUNEL), and for immunohistochemistry.

INTRODUCTIONMonodelphis domestica is the most commonly used laboratory marsupial. In addition to the many factors that make it a convenient laboratory animal (small size, ease of care, nonseasonal breeding), it is the first marsupial whose genome has been sequenced. In this article, we present an overview of aspects of its biology and its use as a model organism. We also discuss basic care, breeding, embryo manipulation, and modifications of common techniques for the study of the development of this species.