CSH protocols最新文献

INTRODUCTIONIt can be useful to assay migration between any two adjacent tissues during development. This protocol assays cell migration between the gonad and mesonephros using tissue recombination between genetically marked and unmarked tissue, combined with an organ culture technique. First, agar blocks are prepared in a custom-built mold. The size and shape of the wells are important to maintain the authentic three-dimensional morphology of the organ; the molds here are designed specifically to accommodate the gonad/mesonephros complex. Freshly dissected organs are then transferred to grooves within the agar blocks, where they are allowed to grow over 24-48 h. Using this protocol, organs develop with good morphology, and show only an ~12-h delay relative to in vivo development.

INTRODUCTIONThe procedure described here provides a quick and reliable method for determining the sex of mouse embryos that are <12.5 days post-coitum (dpc). Cells from amniotic membranes are stained with toluidine blue. The presence of a heavily stained condensed chromatin body (i.e., a Barr body) indicates the XX samples. With experience, we find a >95% concordance with genotyping data based on PCR for Y-chromosome sequences in extracted tail DNA. This protocol has the advantage of speed and efficiency: When assembling cultures with live tissue, samples can be sexed in 30 min.

INTRODUCTIONThe perpetual darkness of caves has two important consequences for permanent inhabitants. First, eyes and pigmentation lose their primary functions. Second, in the absence of photosynthesis, food is rare. For these reasons, cave-adapted species typically have reduced eyes and pigmentation and increased or more efficient metabolisms. Additionally, other senses are usually augmented to compensate for the loss of vision. Identifying the genetic bases underlying these phenotypic changes will enhance our understanding of the specific pathways involved in control of these phenotypes and, in general, the evolutionary process. Unfortunately, the genetics of most cave animals cannot be studied because they are not easily bred. Blind Mexican tetras, Astyanax mexicanus, are the valuable exception to this rule because fish from the various cave populations are fully interfertile with one another and with eyed sister forms still living in nearby surface streams. Hybrids between surface and cave forms permit genetic analysis of their differences, and study of the pure forms as well as of hybrids allows study of their developmental differences. Quantitative trait loci (QTL) analysis has already identified some specific genes responsible for differences between cave and surface forms as well as other likely candidates; more will be added in the future. This system is a valuable addition to the array of existing models for the study of developmental and evolutionary genetics because cave populations are repositories of numerous naturally occurring mutations affecting development of the eyes and other senses, pigmentation, bone structure, metamerism, and metabolism. These alleles have been prescreened by natural selection for high viability, which simplifies their study. In contrast, new alleles obtained through mutagenesis in other model species are typically burdened with lower viability.

INTRODUCTIONCtenophores, or comb jellies, are a group of marine animals whose unique biological features and phylogenetic placement make them a key taxon for understanding animal evolution. Because of its large size, fecundity, abundance in coastal areas, and recent introduction to European waters, Mnemiopsis leidyi (commonly called "sea walnuts") is the most highly studied ctenophore. Under optimal conditions, these self-fertile hermaphrodites are capable of reproduction at 2 wk of age and can release up to 10,000 eggs per day. Adults can be maintained in large aquaria with gentle aeration as long as they are well fed and can be spawned daily; multiple generations can be raised in the laboratory. This protocol describes how to collect embryos from M. leidyi. Under natural conditions, spawning normally occurs ~8 h after sunset, such that eggs are released under the cover of darkness. Because spawning is triggered by the onset of darkness, keeping animals under an artificial light regimen in the laboratory can alter the time of spawning. This protocol is designed to induce animal spawning at approximately 11:00 a.m.; however, it can be adjusted for other times. The duration from spawning to hatching of larvae is 18-24 h.

INTRODUCTIONFunctional proteomics enables protein activities to be studied in vitro using high-throughput (HT) methods. Protein microarrays are the method of choice because they display many proteins simultaneously and require only small reaction volumes to assess function. Protein microarrays are typically used to (1) measure the abundance of many different analytes in a sample or (2) study the functions or properties of many proteins spotted on the array. Target protein microarrays are usually generated by expressing, purifying, and spotting the proteins onto a solid surface at very close spatial density. An alternative approach is to translate the proteins in situ on the array surface. This method uses cell-free extracts that transcribe and translate DNA into proteins which are then captured in situ, thus converting cDNA copies of genes into the desired target proteins. Instead of printing proteins at each feature of the array, the cDNA molecules for the corresponding genes that produce desired proteins are affixed to the array. Chemical treatment of glass slides and DNA isolation can be performed in advance and stored. The plasmid DNA can then be printed to make NAPPA slides, which can be stored dry for use. For experiments, NAPPA slides are expressed followed by detection of proteins and DNA using antibodies and stains. This protocol describes a method for isolating the plasmids in a 96-well format.

INTRODUCTIONCtenophores, or comb jellies, are a group of marine animals whose unique biological features and phylogenetic placement make them a key taxon for understanding animal evolution. Some characteristics are present in nearly all ctenophores, including biradial symmetry, comb rows composed of linked cilia, an apical sensory organ, and two tentacles bearing specialized adhesive cells. All ctenophores studied thus far have the same stereotyped cleavage program and go through a specific stage of development known as the cydippid larva, after which adult structures develop and diverge greatly among species; this is particularly useful for comparative studies. Because of the ease of embryo collection, their size (up to 1 mm in some species), and their rapid development, ctenophores have been attractive animals for experimental embryologists. This protocol describes how to fix ctenophore embryos and their cydippid larvae for antibody staining. Once the samples have been fixed, tissues are incubated with an antibody to the epitope of interest. A secondary antibody conjugated to a fluorescent molecule then reveals the expression pattern of the epitope. Fluorescent microscopy is used to visualize and document the signal. The protocol also includes methods for staining or counterstaining with a fluorescent derivative of phalloidin, which reveals F-actin in muscles and cell borders. Although the protocol focuses on embryonic and larval samples, the technique can also be applied to adult tissues.

INTRODUCTIONCtenophores, or comb jellies, are a group of marine animals whose unique biological features and phylogenetic placement make them a key taxon for understanding animal evolution. Some characteristics are present in nearly all ctenophores, including biradial symmetry, comb rows composed of linked cilia, an apical sensory organ, and two tentacles bearing specialized adhesive cells. All ctenophores studied thus far have the same stereotyped cleavage program and go through a specific stage of development known as the cydippid larva, after which adult structures develop and diverge greatly among species; this is particularly useful for comparative studies. In some cases, gene expression patterns appear to be conserved. Of particular interest is the finding that some genes are expressed in regions of the ctenophore body that are not morphologically distinct from the adjacent areas. However, it has proven difficult to determine the orthology of some genes, possibly because of the extreme divergence of ctenophore representatives. This protocol describes how to isolate total RNA from ctenophore embryos and larvae. After the specimens are sorted, cleaned, and concentrated, they are placed into TRI Reagent, a solution containing phenol and guanidine thiocyanate that allows for the effective isolation of total RNA. The resulting RNA can be used for various applications (e.g., to generate cDNA for reverse transcriptase-polymerase chain reactions). Although the protocol focuses on embryonic and larval samples, the technique can also be applied to adult tissues.

INTRODUCTIONMale and female Astyanax mexicanus can be bred successfully in tanks under appropriate conditions. Females should be maintained on a diet high in fats for 10-14 d before breeding. The transfer of a male and female into clean water in a fresh tank and a change (increase) in water temperature are cues for breeding. Newly fertilized eggs may also be obtained through in vitro fertilization. Note that blind fish should never be paired with eyed fish in illuminated aquaria, because the eyed fish are aggressive and will kill even much larger blind fish. Such matings must be carried out in the dark or by using in vitro fertilization.

INTRODUCTIONFunctional proteomics enables protein activities to be studied in vitro using high-throughput (HT) methods. Protein microarrays are the method of choice because they display many proteins simultaneously and require only small reaction volumes to assess function. Protein microarrays are typically used to (1) measure the abundance of many different analytes in a sample or (2) study the functions or properties of many proteins spotted on the array. Target protein microarrays are usually generated by expressing, purifying, and spotting the proteins onto a solid surface at very close spatial density. An alternative approach is to translate the proteins in situ on the array surface. This method uses cell-free extracts that transcribe and translate DNA into proteins which are then captured in situ, thus converting cDNA copies of genes into the desired target proteins. Instead of printing proteins at each feature of the array, the cDNA molecules for the corresponding genes that produce desired proteins are affixed to the array. Chemical treatment of glass slides and DNA isolation can be performed in advance and stored. The plasmid DNA can then be printed to make NAPPA slides, which can be stored dry for use. For experiments, NAPPA slides are expressed followed by detection of proteins and DNA using antibodies and stains. This protocol describes DNA biotinylation, precipitation, and arraying in preparation for protein expression.

INTRODUCTIONCtenophores, or comb jellies, are a group of marine animals whose unique biological features and phylogenetic placement make them a key taxon for understanding animal evolution. Some characteristics are present in nearly all ctenophores, including biradial symmetry, comb rows composed of linked cilia, an apical sensory organ, and two tentacles bearing specialized adhesive cells. All ctenophores studied thus far have the same stereotyped cleavage program and go through a specific stage of development known as the cydippid larva, after which adult structures develop and diverge greatly among species; this is particularly useful for comparative studies. In some cases, gene expression patterns appear to be conserved. Of particular interest is the finding that some genes are expressed in regions of the ctenophore body that are not morphologically distinct from the adjacent areas. However, it has proven difficult to determine the orthology of some genes, possibly because of the extreme divergence of ctenophore representatives. This protocol describes how to fix, prepare, and hybridize antisense RNA probes in ctenophore embryos and cydippid larvae, as well as how to detect the probes using an alkaline phosphatase-conjugated antibody and colorimetric substrates. Using these techniques, it is possible to determine which cells or tissues express the gene of interest. Although the protocol focuses on embryonic and larval samples, the technique can also be applied to adult tissues.