Showing papers in "CSH Protocols in 2008"

Hematoxylin and Eosin Staining of Tissue and Cell Sections

Abstract: INTRODUCTIONHematoxylin and eosin (H&E) stains have been used for at least a century and are still essential for recognizing various tissue types and the morphologic changes that form the basis of contemporary cancer diagnosis. The stain has been unchanged for many years because it works well with a variety of fixatives and displays a broad range of cytoplasmic, nuclear, and extracellular matrix features. Hematoxylin has a deep blue-purple color and stains nucleic acids by a complex, incompletely understood reaction. Eosin is pink and stains proteins nonspecifically. In a typical tissue, nuclei are stained blue, whereas the cytoplasm and extracellular matrix have varying degrees of pink staining. Well-fixed cells show considerable intranuclear detail. Nuclei show varying cell-type- and cancer-type-specific patterns of condensation of heterochromatin (hematoxylin staining) that are diagnostically very important. Nucleoli stain with eosin. If abundant polyribosomes are present, the cytoplasm will have a distinct blue cast. The Golgi zone can be tentatively identified by the absence of staining in a region next to the nucleus. Thus, the stain discloses abundant structural information, with specific functional implications. A limitation of hematoxylin staining is that it is incompatible with immunofluorescence. It is useful, however, to stain one serial paraffin section from a tissue in which immunofluorescence will be performed. Hematoxylin, generally without eosin, is useful as a counterstain for many immunohistochemical or hybridization procedures that use colorimetric substrates (such as alkaline phosphatase or peroxidase). This protocol describes H&E staining of tissue and cell sections.

Bone Marrow-Derived Macrophages (BMM): Isolation and Applications

Abstract: 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.

In Planta Visualization of Protein Interactions Using Bimolecular Fluorescence Complementation (BiFC).

Abstract: INTRODUCTIONBimolecular fluorescence complementation (BiFC) analysis enables direct visualization of protein-protein interactions in living cells. This method has been successfully adapted to a variety of expression systems in different organisms. BiFC is based on the formation of a fluorescent complex by fragments of the enhanced yellow fluorescent protein (eYFP) when brought together by the interaction of two associating proteins fused to these fragments. Interaction of these proteins restores fluorescence and allows the visualization of spatial localization patterns of protein complexes. Absence of interaction prevents reassembly of the fluorescent protein and results only in background fluorescence. The specificity of bimolecular fluorescence complementation must be confirmed by parallel analysis of proteins in which the interaction interface has been mutated. This protocol describes the Agrobacterium-mediated transient expression protocol for BiFC assays in Nicotiana benthamiana leaf cells. This method exhibits a high transformation rate (up to 90% of the cells) and allows the simultaneous expression of multiple proteins in single cells. Therefore, this expression system enables colocalization analyses of fluorescently labeled proteins with the formation of BiFC complexes for determination of cellular complex localization. In addition, protein interaction assays in N. benthamiana leaves permit the investigation of protein interactions at different time points of expression, allow analysis of proteins that are normally toxic in protoplasts, and enable comparative protein interaction investigation in epidermal cells as well as in mesophyll protoplasts.

Tomato (Solanum lycopersicum): A Model Fruit-Bearing Crop.

Abstract: INTRODUCTIONTomato (Solanum lycopersicum) is one of the most important vegetable plants in the world. It originated in western South America, and domestication is thought to have occurred in Central America. Because of its importance as food, tomato has been bred to improve productivity, fruit quality, and resistance to biotic and abiotic stresses. Tomato has been widely used not only as food, but also as research material. The tomato plant has many interesting features such as fleshy fruit, a sympodial shoot, and compound leaves, which other model plants (e.g., rice and Arabidopsis) do not have. Most of these traits are agronomically important and cannot be studied using other model plant systems. There are 13 recognized wild tomato species that display a great variety of phenotypes and can be crossed with the cultivated tomato. These wild tomatoes are important for breeding, as sources of desirable traits, and for evolutionary studies. Current progress on the tomato genome sequencing project has generated useful information to help in the study of tomato. In addition, the tomato belongs to the extremely large family Solanaceae and is closely related to many commercially important plants such as potato, eggplant, peppers, tobacco, and petunias. Knowledge obtained from studies conducted on tomato can be easily applied to these plants, which makes tomato important research material. Because of these facts, tomato serves as a model organism for the family Solanaceae and, specifically, for fleshy-fruited plants.

The dogfish Scyliorhinus canicula: A reference in jawed vertebrates

Abstract: 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.

Cultures of Cerebellar Granule Neurons

Abstract: 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.

Strategies for Overcoming PCR Inhibition

Abstract: INTRODUCTIONThe use of conventional and real-time PCR is to some extent restricted by the presence of PCR inhibitors. This is particularly so when the techniques are applied directly to complex biological samples such as clinical, environmental, or food samples for the detection of microorganisms. PCR inhibitors can originate from the sample itself, or as a result of the method used to collect or otherwise prepare the sample. Either way, inhibitors can dramatically reduce the sensitivity and amplification efficiency of PCR. This article discusses methods of reducing inhibition and designing reliable and sensitive conventional and real-time PCR experiments.

The Two-Spotted Cricket Gryllus bimaculatus: An Emerging Model for Developmental and Regeneration Studies

Abstract: 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.

Paraffin embedding tissue samples for sectioning.

Abstract: INTRODUCTIONThis protocol describes a method for embedding tissues in paraffin blocks for sectioning. Paraffin sections require extensive fixation and processing steps, but provide superior morphology compared with other sectioning methods.

Establishing and maintaining a colony of planarians.

Abstract: 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.

Transfection of HEK293-EBNA1 Cells in Suspension with Linear PEI for Production of Recombinant Proteins

Abstract: INTRODUCTIONFast and efficient production of recombinant proteins (r-proteins) remains a major challenge for the academic and biopharmaceutical communities. Pure r-proteins are often required in large amounts (hundreds of milligrams to gram quantities) when being developed as biotherapeutics, or in smaller quantities (milligrams) for high-throughput screening campaigns and structural or functional studies. Mammalian cells are often preferred over prokaryotic systems when expressing cDNAs of mammalian origin due to their superior capability to conduct elaborate post-translational modifications. Large-scale transfection of mammalian cells is now establishing itself as a "must-have" technology in the scientific community, as it allows the production of milligram to gram quantities of r-proteins within a few days after cDNA cloning into the appropriate expression vector. Although calcium-mediated large-scale transfection is very effective, it is usually achieved in serum-containing medium under tightly controlled conditions that are difficult to achieve on a large scale. In contrast, polyethylenimine (PEI) is much easier to use: It binds to and precipitates DNA efficiently and the resulting DNA-PEI complexes are suitable for efficient transfection of mammalian cells. PEI has been used successfully on a large scale in serum-containing and serum-free cultures. In particular, the linear isoform of PEI is more effective for transfecting cells in suspension. This protocol describes the steps needed for successful transfection of HEK293 cells adapted to serum-supplemented or serum-free medium in suspension culture using linear PEI.

Phenotypic Analysis of Arabidopsis Mutants: Trypan Blue Stain for Fungi, Oomycetes, and Dead Plant Cells

Abstract: INTRODUCTIONTrypan blue stains vasculature, dead plant cells, and fungal and oomycete hyphae. It is useful for assessing the extent of colonization of tissue, and for detecting microlesions present in certain lesion-mimic mutants. Trypan blue staining requires chloral hydrate for destaining, which is inconvenient, because it is a controlled substance. The chloral hydrate can be replaced with 1:2 lactophenol:ethanol, but the background staining will be higher than it is when chloral hydrate is used.

Live Imaging of Planarian Membrane Potential Using DiBAC4(3)

Abstract: INTRODUCTIONThis protocol describes how to use the anionic membrane voltage-reporting dye DiBAC(4)(3) to generate images of cell membrane potential in live planarians. These images qualitatively reveal variations in time-averaged membrane potential across different regions of the organism. Changes in these images due to experimental treatments reveal how the particular treatment affects this physiological parameter. This method is a great improvement over standard electrophysiological techniques, which cannot be used to gain an understanding of the electrical properties of an entire worm or a regenerating fragment, due to small cell size and large cell number. When the proper controls are performed, this technique is a very powerful and simple way to gather physiologic data.

Whole-mount fluorescence immunocytochemistry on Xenopus embryos.

Abstract: INTRODUCTIONImmunocytochemistry (ICC) is widely exploited in studying mammalian systems, but is underutilized among Xenopus developmental biologists. This stems, in part, from the relatively small number of Xenopus antibodies available for use in research. Common misconceptions about ICC in Xenopus embryos also prevail, discouraging researchers from trying the procedure. However, ICC with Xenopus is simple and effective. This article describes methods for whole-mount ICC in Xenopus embryos. Also included are simple procedures to quench autofluorescence of Xenopus and to remove surface pigment from embryos which may interfere with fluorescence imaging. The methods described here are useful for detecting tissue-specific probes (e.g., 12/101 to detect somites). They are also effective for imaging the cytoskeleton (e.g., α-tubulin to detect microtubules) or localizing specific proteins at the subcellular level (e.g., ZO-1 to detect tight junctions). In addition, combining ICC with in situ hybridization is simple and highly effective.

PCR Mutagenesis by Overlap Extension and Gene SOE

Abstract: INTRODUCTIONMutagenesis by PCR is accomplished by incorporating desired genetic changes into custom-made primers used in amplification reactions. Because these mutagenizing primers have terminal complementarity, two separate DNA fragments amplified from a target gene can be fused into a single product by primer extension without relying on restriction endonuclease sites or ligation reactions. Briefly, mutagenesis is achieved by performing PCR with specially designed oligonucleotide primers that include the desired substitutions, insertions, or deletions in their sequence. The two overlapping fragments are fused together in a subsequent extension reaction. The inclusion of outside primers in the extension reaction amplifies the fused product by PCR. Theoretically, the primers can be moved anywhere along the targeted gene to introduce mutations. This method can be exploited further by using DNA fragments from different sources. Such gene splicing by overlap extension (SOE) can be used to rapidly produce chimeras. A limitation of SOE is the difficulty of manipulating large DNA segments (i.e., >1-2 kb). To circumvent this, a cassette system can be targeted, modified by SOE, and reinserted using restriction endonuclease sites designed into the cassette structure. This approach also allows easy shuffling or replacement of gene segments.

Coimmunoprecipitation (co-IP) of Nuclear Proteins and Chromatin Immunoprecipitation (ChIP) from Arabidopsis.

Abstract: INTRODUCTIONTranscriptional reprogramming occurs during development and in response to diverse stimuli and stresses. The isolation and characterization of nuclear proteins, particularly those binding to DNA and chromatin, are therefore important to understanding these processes. Two specific approaches to understanding the function of nuclear proteins involve the characterization of their protein-protein interactions, and of the transcriptional targets of specific transcription factors. Coimmunoprecipitation (co-IP) is a straightforward technique to study in vivo protein-protein interactions, and can identify interacting proteins or protein complexes present in cell extracts. Chromatin immunoprecipitation (ChIP) permits the identification of protein-DNA interactions in pull-down assays using specific antibodies against DNA-binding proteins, such as transcription factors or histone/chromatin-binding proteins. Here, we present detailed protocols for extraction of Arabidopsis seedlings, co-IP of nuclear proteins, and ChIP.

The nissl stain: a stain for cell bodies in brain sections.

Abstract: INTRODUCTIONThe Nissl stain is used widely in many research labs to examine the overall morphology of the brain. It is often used to verify the location of a lesion or an electrode.

Using BLOSUM in Sequence Alignments.

Abstract: INTRODUCTIONThe original Dayhoff percent accepted mutation (PAM) matrices were developed based on a small number of protein sequences and an evolutionary model of protein change. By extrapolating from the observed changes at small evolutionary distances to large ones, it was possible to establish a PAM250 scoring matrix for sequences that were highly divergent. Another approach to finding a scoring matrix for divergent sequences is to start with a more divergent set of sequences and produce a scoring matrix from the substitutions found in those less-related sequences. The blocks amino acid substitution matrices (BLOSUM) scoring matrices were prepared this way. This article explains how BLOSUM scoring matrices were created and how they can best be used.

Preparation of Slides and Coverslips for Microscopy

Abstract: INTRODUCTIONIt is imperative that the slides and coverslips used in fluorescence microscopy procedures be extremely clean. Although coverslips look clean, especially when a new box is first opened, they may have a thin film of grease on them that will not allow tissue culture cells to adhere well and that may interfere with some processing steps in certain protocols. Therefore, coverslips should routinely be washed with acid or base solutions to rid them of this film. Commercial precleaned slides are also likely to be dirty and must be washed prior to use. This protocol describes various approaches for cleaning slides and coverslips and sterilizing them for cell culture, as well as methods for subbing slides. In the subbing procedure, slides are coated with gelatin, aminoalkylsilane, or poly-L-lysine solution to promote the adhesion of cells or tissues to the glass surface. Gelatin or aminoalkylsilane is usually used for tissue sections or small organisms, whereas poly-L-lysine is routinely used for cultured cells.

Planarians: a versatile and powerful model system for molecular studies of regeneration, adult stem cell regulation, aging, and behavior.

Abstract: INTRODUCTIONIn recent years, planarians have been increasingly recognized as an emerging model organism amenable to molecular genetic techniques aimed at understanding complex biological tasks commonly observed among metazoans. Growing evidence suggests that this model organism is uniquely poised to inform us about the mechanisms of tissue regeneration, stem cell regulation, tissue turnover, pharmacological action of diverse drugs, cancer, and aging. This article provides an overview of the planarian model system with special attention to the species Schmidtea mediterranea. Additionally, information is provided about the most popular use of this organism, together with modern genomic resources and technical approaches.

Comb jellies (ctenophora): a model for Basal metazoan evolution and development.

Abstract: 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.

Whole-Mount In Situ Hybridization of Spider Embryos

Abstract: 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 detection of transcripts in fixed C. salei embryos using whole-mount in situ hybridization.

Comparison of the PAM and BLOSUM Amino Acid Substitution Matrices.

Abstract: INTRODUCTIONThe choice of a scoring system including scores for matches, mismatches, substitutions, insertions, and deletions influences the alignment of both DNA and protein sequences. To score matches and mismatches in alignments of proteins, it is necessary to know how often one amino acid is substituted for another in related proteins. Percent accepted mutation (PAM) matrices list the likelihood of change from one amino acid to another in homologous protein sequences during evolution and thus are focused on tracking the evolutionary origins of proteins. In contrast, the blocks amino acid substitution matrices (BLOSUM) are based on scoring substitutions found over a range of evolutionary periods. There are important differences in the ways that the PAM and BLOSUM scoring matrices were derived. These differences, which are discussed in this article, should be appreciated when interpreting the results of protein sequence alignments obtained with these matrices.

Whole-Genome Amplification by Degenerate Oligonucleotide Primed PCR (DOP-PCR).

Abstract: INTRODUCTIONPCR-based whole-genome amplification (WGA) has the goal of generating microgram quantities of genome-representative DNA from picogram or nanogram amounts of starting material. This amplification should introduce little, or ideally no, representational bias. Unlike other techniques for WGA, PCR-based methods are generally less affected by DNA quality and are more applicable to DNA extracted from various sources (fixed and fresh tissues). The degenerate-oligonucleotide-primed PCR (DOP-PCR) method described here allows complete genome coverage in a single reaction. In contrast to the pairs of target-specific primer sequences used in traditional PCR, only a single primer, which has defined sequences at its 5'-end (containing an XhoI restriction site) and 3'-end and a random hexamer sequence between them, is used here. DOP-PCR comprises two different cycling stages. In stage 1 (low stringency), low-temperature annealing and extension in the first five to eight cycles occurs at many binding sites in the genome. The 3'-end of the primer binds at sites in the genome complementary to the 6-bp well-defined sequence at the 3'-end of the primer (~10(6) sites in the human genome). The adjacent random hexamer sequence (displaying all possible combinations of the nucleotides A, G, C, and T) can then anneal and tags these sequences with the DOP primer. In stage 2 (high stringency; >25 cycles), the PCR annealing temperature is raised, which increases priming specificity during amplification of the tagged sequence. DOP-PCR generates a smear of DNA fragments (200-1000 bp) that are visible on an agarose gel.

Opossum (Monodelphis domestica): A Marsupial Development Model.

Abstract: 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.

The Demosponge Amphimedon queenslandica: Reconstructing the Ancestral Metazoan Genome and Deciphering the Origin of Animal Multicellularity.

Abstract: 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.

The GAL4 System: A Versatile Toolkit for Gene Expression in Drosophila

Abstract: INTRODUCTIONThe generation of gain-of-function phenotypes by ectopic expression of known genes provides a powerful complement to the genetic approach, in which genes are studied or identified through mutations that generally reduce or eliminate gene function. The GAL4 system is a method for ectopic gene expression that allows the selective activation of any cloned gene in a wide variety of tissue- and cell-specific patterns. A key advantage of the system is the separation of the GAL4 protein from its target gene in distinct transgenic lines, which ensures that the target gene is silent until the introduction of GAL4. Recent modifications and adaptations of the GAL4 system to make the system inducible have further expanded its scope, enabling greater temporal control over the activity of GAL4. There are now large resources for the community, including thousands of GAL4 lines and a wide selection of reporter lines. Here we present an overview of the GAL4 system, highlighting recent developments and discussing methods for generating and analyzing transgenic flies for GAL4-mediated ectopic expression.

Isolation of amphimedon developmental material.

Abstract: 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.

Copy Number Variation Detection via High-Density SNP Genotyping.

Abstract: INTRODUCTIONHigh-density single nucleotide polymorphism (SNP) genotyping arrays recently have been used for copy number variation (CNV) detection and analysis, because the arrays can serve a dual role for SNP- and CNV-based association studies. They also can provide considerably higher precision and resolution than traditional techniques. Here we describe PennCNV, a computational protocol designed for CNV detection from high-density SNP genotyping data. This protocol extracts allele-specific signal intensities from genotyping arrays, and then integrates information on SNP spacing and SNP allele frequencies to generate CNV calls by a hidden Markov model (HMM) algorithm. Analyses of CNVs from SNP genotyping arrays will provide a more comprehensive view of genome variation, and complement current genome-wide association studies in identifying disease susceptibility loci.

Immunohistochemistry of Whole-Mount Mouse Embryos

Abstract: INTRODUCTIONThe technique presented this protocol is ideal for obtaining an overall, three-dimensional picture of the distribution of an antigen in embryos from the prestreak stage to ~10.5 dpc (days post-coitum). The embryos are fixed and permeabilized so that antibodies can penetrate all of the tissues. However, antibodies will not penetrate the embryo completely if it is too large, and so it is better to dissect specific organs (e.g., brain, lungs, and gut) and process them separately.