The most commonly used bacterial pathogen of Arabidopsis is Pseudomonas syringae, pathovar tomato strain DC3000 or pathovar maculicola strain ES4326. Plants used for infection with P. syringae should be grown on a short-day light cycle, so that they develop large leaves. For consistent results, it is important that the plants are watered well and do not experience any abiotic stresses. The plants should be grown and tested in a temperature- and humidity-controlled growth chamber, because the extent of bacterial growth is highly dependent on both factors. Gene-for-gene resistance to P. syringae is usually accompanied by the hypersensitive response (HR), a form of localized cell death that occurs in response to pathogen challenge. P. syringae strains carrying one of the avirulence genes avrRpt2, avrRpm1, avrB, avrPphB, and avrRps4 trigger the HR in wild-type Columbia. Other ecotypes may lack one or more of the R genes needed for recognition of these avirulence genes. Bacterial growth can be monitored by grinding up infected tissue, plating serial dilutions on King’s B medium, and counting colonies. This protocol describes methods for preparing bacterial cultures, inoculating plants, testing the HR, and assessing bacterial growth.

This protocol describes how to incubate painted turtle eggs and collect embryos for gene expression analysis. The same basic protocol can be used to incubate eggs and collect embryos from other turtle species by modifying the incubation temperature to suit the particular developmental needs of each species.

Because of the great diversity of ants, it is difficult to give a single protocol for the collection of live specimens. Ant body size can be very small or extremely large; the ants can be hard or soft, sting or spray toxic chemicals, live in the open or in hard-to-reach places; and colony size can range from tens of individuals to millions. Thus, collection techniques must be tailored to each particular species. In particular, caution must always be taken when dealing with stinging species, and symptoms and basic first-aid measures, especially for the treatment of anaphylactic shock, should be reviewed before beginning fieldwork. Nonetheless, many species are collectable as whole colonies. This protocol reviews some basic techniques for collecting ground-nesting species and describes how to collect whole live colonies (with queens), which are necessary for long-term laboratory studies and addressing questions of social organization and ecology.

Ant societies are tractable and malleable, two features that make them ideal models for probing the organization of complex biological systems. The ability to identify specific individuals while they function as part of a colony permits an integrative analysis of social complexity, including self-organizational processes (i.e., how individual-level properties and social interactions give rise to emergent, colony-level attributes such as division of labor and collective decision making). Effects of genotype, nutrition, and physiology on individual behavior and the organization of work also can be investigated in this manner, through correlative and manipulative approaches. Moreover, aspects of colony demography (e.g., colony size, and age and size distributions of workers) can be altered experimentally to examine colony development and regulatory mechanisms underlying colony homeostasis and resiliency. This protocol describes how to sample the behavior of ants living in a colony under laboratory conditions. Specifically, it outlines how to identify and observe individuals within a colony, an approach that can be used to quantify individual- and colony-level patterns of behavior. When a lower-resolution measure of overall group behavior is desired, individual identities might not be required. Given the diversity of ants and their study, this protocol provides a very general methodology; the details can be modified according to the body size, colony size, and ecology of the focal species, as well as to specific research aims. These basic techniques can also be extended to more advanced experimental designs such as manipulation of colony demography and hormone treatment.

Ants are among the most dominant taxa in terrestrial ecosystems, despite their small individual size. Furthermore, they are a hyperdiverse family with an estimated 20,000 species. Together, these two properties make ants a model for ecological interactions (specifically competition) and biodiversity estimation. Although there are many means of measuring diversity, the two most common among myrmecologists are baiting and pitfall trapping. Pitfall traps provide an almost unbiased estimate of the ground foraging community, whereas baiting allows the estimation of ecological dominance and the competitive outcomes between species. This protocol describes an approach to assay both abundance (pitfall traps) and ecological interactions (baits) in the same community.

Over the past 20 yr, the use of stable isotopes to infer feeding ecology and the examination of how energetic and elemental exchanges are affected by and affect life (ecological stoichiometry) have gained momentum. The ecological diversity of ants makes them interesting models to explore dietary ecology and their role in food webs. Moreover, their ecological dominance in most habitats facilitates sampling. The protocol described here will produce samples adequate for submission to most labs that specialize in high-throughput analysis of stable isotopes; one should check with any particular lab for specific submission instructions. Note, however, that this protocol is designed specifically for the quantification of the natural abundance of stable isotopes; it does not cover the preparation of trace samples.

Stored fat can be informative about the relative age of an ant, its nutritional status, and the nutritional status of the colony. Several methods are available for the quantification of stored fat. Before starting a project involving fat extraction, investigators should weigh the advantages and disadvantages of different methods in order to choose the one that is best suited to the question being addressed. This protocol, although not as accurate as some alternatives, facilitates the rapid quantification of many individuals.

Dissection of the reproductive system of ant workers and queens can be useful for answering many questions. Observations of ovarian status in both female castes can be used to identify relationships between other factors and the ovaries, determine whether an individual has laid eggs, and, with more advanced molecular techniques, identify the end product of the oocyte in question (e.g., trophic egg, viable egg, nonviable egg). In addition, dissection of queens allows for observation of the spermatheca (the sperm storage organ), and thus identification of whether an individual has mated. Sperm can also be sampled for genetic analyses. In this protocol, we describe the dissection of the female ant reproductive system. We also discuss dissection of the corpora allata glands, where juvenile hormone is produced.

Many different DNA isolation methods have been employed successfully in ants. Parameters such as the size and developmental stage of the specimen (egg, larvae, or adult) and the subsequent use of the DNA will mostly determine which method should be used. Ant body sizes range from minute (1-2 mm in length) to large (30 mm), and the volume of the initial digestion should be adjusted accordingly. Whereas workers usually have low concentrations of storage proteins and fat, queens and larvae can contain considerable amounts of these substances that can interfere with the subsequent use of the isolated DNA. Ants also have many glands in the head and abdomen, and the contents of these glands can also interfere with the successful application of polymerase chain reaction (PCR) or restriction digests of the isolated DNA. This protocol presents two DNA isolation methods that have worked reliably for a wide range of ant species: a "quick and dirty" technique using Chelex isolation, and a more elaborate, classical phenol:chloroform procedure.

Juvenile hormone (JH) is an important insect hormone known to have many effects on development, reproduction, and behavior in both solitary and social insects. A number of questions using ants as a model involve JH. This procedure allows for quantification of circulating levels of JH III, which can be an important factor in many questions relating to insect research. The JH III is extracted from a subject, purified, and converted to a d₃-methoxyhydrin derivative that can be quantified by gas chromatography-mass spectrometry (GC-MS). The major advantages of this protocol are its high resolution, and its ability to quantify significant differences between relatively small quantities of the hormone. Its major limitations are the time necessary to process samples, its relatively high cost, and maintaining the sensitivity of the equipment.

Ecdysteroids are a group of steroid compounds present in many plant and invertebrate species. In arthropods, they function primarily as hormones involved in the regulation of molting. This protocol describes how to extract ecdysteroid hormones from ant specimens and subsequently quantify circulating levels of the hormone. The hormone can be extracted from hemolymph or from whole-body homogenates of insects and quantified by radioimmunoassay.

Juvenile hormone (JH) is an important insect hormone known to have many effects on development, reproduction, and behavior in both solitary and social insects. This protocol describes how to quantify in vitro biosynthesis rates from excised corpora allata (CA), the glands responsible for JH production. Excised glands are incubated with radiolabeled methionine, resulting in the production of radiolabeled JH III, which can then be quantified. This protocol is most useful when quantification of the activity of the glands, rather than circulating JH levels, is desired. Its difficulties lie primarily in the ability to remove and handle the glands correctly.

Living in a predominantly dark environment, ants rely mostly on chemical signals for communication. Trail and alarm pheromones are the most widely studied and best characterized of all ant semiochemicals, but other such compounds can influence a variety of other behaviors, including reproductive activities, sexual development, nest mate and caste recognition, and defense. A typical worker body contains more than 10 different semiochemical-producing glands, and the surface of the cuticle is covered with lipids that serve as recognition signals. The methods of choice for collection and identification of ant semiochemicals should be determined based on results of behavioral analyses. These can indicate the source (e.g., glandular, cuticular) and the nature (volatile vs. nonvolatile) of the chemical. This protocol presents a number of different methods for collecting lipid semiochemicals. These can be followed by gas chromatography (GC) coupled with mass spectrometry (MS) to better characterize, and possibly identify, the semiochemical in question.

The detection of transcript distribution throughout a fixed tissue is a major step in studying the transcriptional activity of target genes and their function. In situ hybridization specifically detects the spatial distribution of RNA transcripts using an antisense RNA probe. This protocol describes the preparation of digoxigenin-labeled antisense RNA probes and their hybridization to complementary mRNA sequences; expression can then be localized using an antibody against digoxigenin conjugated to a chromogenic enzyme. In ants, this method can be applied to visualize cell populations of interest among other populations in a tissue, such as insect ovaries, or in the whole organism, such as insect embryos. Specific markers (e.g., genes known to be expressed in particular clusters of cells) can be cloned and used as probes to study the distribution and development of germline cells (e.g., nanos, vasa), neurons (repo), or limb structures (e.g., distal-less). Various markers might also allow the study of oogenesis (nanos, par-1, oskar), segmentation in the embryo (e.g., engrailed, wingless), or other developmental processes.

To create a genetic linkage map of an ant genome, the phase of a marker (i.e., which marker/allele came from the grandmother and which came from the grandfather of the individuals used for linkage mapping) must be known. However, field colonies contain only two generations: the queen(s) and their offspring. Normally, virgin queens disperse from their nest, mate with one or multiple males who die after mating, and start a new colony. A new reproductive brood (i.e., virgin queens and males) is usually produced after colony establishment, which can take from 6 mo to 3-5 yr. Hence, determining the phase of markers from field samples is impossible because the grandparents of a reproductive queen are no longer available for genetic analysis. Ants raised in the laboratory are likewise unsuitable for such analyses, because most ants cannot be bred regularly in the laboratory, and attempts to inseminate ant queens artificially have not been successful. However, three features facilitate "phase-unknown" linkage mapping for any ant (or other social hymenopteran) species, which is described here: (1) Mature colonies usually produce many reproductive offspring during a short period of time, which allows for the collection of a good-sized mapping population (>100 males) from a field colony; (2) Ants have a haplodiploid sex determination system (i.e., males are haploid and females diploid); (3) Queens produce males parthenogenetically. Thus, because they have no father, only those markers for which the queen is heterozygous are expected to segregate 1:1 in her male offspring.

Nested Patch polymerase chain reaction (PCR) amplifies a large number (greater than 90) of targeted loci from genomic DNA simultaneously in the same reaction. These amplified loci can then be sequenced on a second-generation sequencing machine to detect single nucleotide polymorphisms (SNPs) and mutations. The reaction is highly specific: 90% of sequencing reads match targeted loci. Nested Patch PCR can be performed on many samples in parallel, and by using sample-specific DNA barcodes, these can be pooled and sequenced in a single reaction. Thus, the Nested Patch PCR protocol that is described here provides an easy workflow to identify SNPs and mutations across many targeted loci for many samples in parallel.

Conventional cultivation-based methods to measure microbial abundance are unsuitable for quantifying uncultured microorganisms that constitute the majority of microbial life in most environmental or medical samples. This problem is solved by the quantification approach described here, which combines fluorescence in situ hybridization (FISH) with rRNA-targeted probes and digital image analysis. By measuring the areas of probe-labeled biomass in randomly recorded image pairs, an unbiased estimate of the relative biovolume of the population of interest can be obtained. This approach expresses abundance as "biovolume fraction" (relative to the total biovolume of the whole microbial community). This value equals the share of biochemical reaction space occupied by the quantified population and thus can be more relevant ecologically than absolute cell numbers (e.g., a few large cells can contain the same biovolume as many small cells). Another advantage lies in the complete independence of this method from the morphology of the quantified organisms. Regardless of whether the target microbes occur as single cells in plankton samples, as filaments, or as dense aggregates in biofilms, this cultivation-independent method allows the composition of complex microbial communities to be determined.

Standard methods for extracting DNA from cells or organisms (e.g., phenol extraction and ethanol precipitation) produce fragments with an average size of 50-200 kb under optimal conditions. The shearing forces that are applied to DNA in solution during mechanical vortexing or mixing and pipetting produce frequent double-stranded breaks. To prepare high-molecular-weight (HMW) DNA, it is necessary to guard against such damaging forces by performing all extractions and manipulations on DNA that is embedded within a protective matrix. Preparation of HMW DNA from Drosophila embryos is described in detail here because, in our hands, it is the simplest and most reliable protocol and can be used for large- or small-scale preparations. The overall strategy is to purify nuclei, gently embed them in molten agarose, and then extract proteins and perform other enzymatic reactions by transferring the solidified agarose block into the appropriate solutions. Salts, soaps, and enzymes act on the DNA by diffusing through the agarose matrix, while the matrix protects the DNA from shearing forces.

This protocol describes a simple, fast, and efficient method for making adhesive micropatterns that can be used to control individual cell shape and adhesion patterns. It is based on the use of an elastomeric stamp containing microfeatures to print proteins on the substrate of choice. The process can be subdivided into three parts. First, a silicon master is fabricated, which contains the microfeatures of interest. Once fabricated, the master can be used multiple times to make stamps. Masters with customized patterns can also be purchased commercially. Second, a polydimethylsiloxane (PDMS) stamp is fabricated. Unlike fabrication of the master, this step can be performed without specialized equipment. The PDMS stamp is inked with extracellular matrix proteins. Proteins are printed on a substrate (e.g., a tissue culture polystyrene dish or a glass coverslip covered with a thin layer of polystyrene). The nonprinted areas are back-filled with poly-L-lysine-polyethylene glycol, which renders them resistant to cell adhesion. The production of these micropatterned substrates can be completed in <2 h. The third and final portion of the protocol describes the deposition of cells onto the micropatterned substrate.

Aneuploidy in embryonic stem (ES) cells is the major cause of failure in obtaining contributions to all tissues of chimeras, including the germ line. This protocol describes a simple method for counting chromosomes from metaphase spreads in ES cells. ES cell clones showing euploidy on 70%-80% of spreads are used for chimera production.

The eukaryotic cell has evolved to compartmentalize its functions and transport various metabolites among cellular compartments. Therefore, it is important to study cellular organization and structure/function relationships. The mitochondrion is a double-membraned organelle that is important in various metabolic processes, including oxidative phosphorylation, electron transport, the Krebs cycle, β-oxidation of fatty acids, and part of the urea cycle. This protocol presents methods for labeling mitochondria in both live and fixed cells using several different fluorescent molecules. Rhodamine 123, tetramethylrhodamine ethyl ester (TMRE), and tetramethylrhodamine methyl ester (TMRM) are membrane-potential-sensitive, cationic fluorophores. Therefore, the mitochondrion must be functioning and generating a membrane potential in order to attract and maintain the dyes in the mitochondrion. These dyes are also used as sensitive indicators of the mitochondrial membrane potential. Rhodamine 123 is specific for the mitochondrion. In contrast, TMRM, TMRE, and the carbocyanine dyes can also label the endoplasmic reticulum to some degree. Mito Tracker CMTMRos has the added feature of chemical reactivity so that, once incorporated in the mitochondrion, it can chemically link to thiol groups and will not leave the mitochondrion when the membrane potential decreases as a result of fixation and/or cell death. Hence, it can be used to locate mitochondria in specimens that will subsequently be fixed. The effect of submicromolar concentrations of mitochondrial inhibitors, for example, KCN, rotenone, antimycin , and uncouplers of oxidative phosphorylation, such as carbonylcyanide-m-chlorophenyl hydrazone (CCCP) and the related carbonylcyanide-p-trifluoromethoxyphenyl hydrazone (FCCP), can be examined on isolated mitochondria or mitochondria in intact cells.

The oxidative burst characteristic of gene-for-gene resistance responses and early events in establishment of systemic acquired resistance can be detected using 3,3'-diaminobenzidine tetrahydrochloride (DAB), a stain for hydrogen peroxide. The presence of hydrogen peroxide, which is symptomatic of the hypersensitive response, causes polymerization of DAB, yielding a brown color. This protocol describes how to stain Arabidopsis tissue with DAB.

The Wnt/β-catenin pathway is the best characterized Wnt pathway largely as a result of powerful assays that allow measurement of in vivo and in vitro pathway activation. Early assays for Wnt/β-catenin signaling included phenotypic assays in Drosophila, dorsal axis duplication in Xenopus, and proliferation of C57MG mammary epithelial cells. More recent characterizations rely on direct measurement of pathway activation. The Wnt/β-catenin signaling pathway culminates with T-cell factor/lymphoid enhancer factor (TCF/LEF)-dependent regulation of target gene transcription; assaying such transcription with reporters containing multimerized TCF/LEF DNA-binding sites is a reliable measure of pathway activation. These reporters have helped characterize the Wnt/β-catenin signaling pathway and will be critical to further studies of pathway regulation and in the development of therapeutic mechanisms to combat diseases arising from aberrant signaling. This protocol describes a variety of methods for using an improved Wnt/β-catenin reporter system, the β-catenin-activated reporter (BAR), and its accompanying control reporter system, found-unresponsive BAR (fuBAR). Procedures are presented for transient and stable transfection of the reporter. Transient transfection is very robust and does not require lentiviral production, although it has a slightly smaller dynamic range relative to stably integrated BAR. The stable transduction system can be used to generate stable reporter cell lines, assay Wnt/β-catenin signaling in cells that might be otherwise difficult to transfect, or assay signaling in vivo. The BAR system, with its improved sensitivity, greater dynamic range, and lentiviral-based reporter system, provides a vital tool for future studies of this signaling pathway.

This protocol describes the preparation and fixation of honeybee tissue. Honeybee embryos are very fragile and must be fixed overnight before they can be subjected to downstream applications such as in situ hybridization and immunohistochemistry.

This protocol outlines how to perform in situ hybridization on honeybee embryos, adult brains, or ovaries to detect the expression pattern of a targeted transcript. The tissue is digested by proteinase K to promote probe penetration, and then prehybridized to block nonspecific binding of the probe.

This protocol outlines how to carry out section in situ hybridization on honeybee larvae to detect expression of a gene of interest in individual cell types. It can also be modified to investigate gene expression in a sectioned honeybee brain. A digoxigenin-labeled probe is hybridized to fixed sections and the probe is detected by 4-nitro blue tetrazolium chloride (NBT)-5-bromo-4-chloro-3-indolyl-phosphate (BCIP) color development.

This protocol describes the preparation and staining of honeybee embryos for immunohistochemistry. An antigen retrieval step is required in order to restore antigens that have become masked by the long fixation step required for honeybee embryos.

This protocol describes RNA interference (RNAi)-mediated knockdown of a target gene in honeybee embryos. dsRNA is directly injected into freshly laid embryos in order to disrupt the function of a specific gene during honeybee embryogenesis.

This protocol describes the extraction of DNA from forensic samples using Chelex, a chelating ion exchange resin suspension.

Magnetofection is defined as nucleic acid delivery guided and mediated by magnetic force acting on associations of magnetic particles and nucleic acids or nucleic acid vectors. Vectors are bound to magnetic, usually iron oxide, nanoparticles, in most cases by noncovalent bonds. Magnetic force accumulates and/or holds magnetic vectors in a target tissue against hydrodynamic forces. In cell culture, magnetic vectors are magnetically sedimented on the target cells within minutes. Thus, the diffusion barrier to nucleic acid delivery is overcome, the full vector dose comes in contact with the target cells, and introduction of genetic material is synchronized. Nucleic acid delivery is greatly accelerated and its efficiency with many, if not most, vector types is improved. Magnetofection is applicable to small and large nucleic acids. Other advantages include low-dose requirements, the possibility of confining nucleic acid introduction to a localized area (magnetic targeting), and the amenability to high-throughput automation. Due to the favorable dose-response profile and the rapid kinetics, vector-related toxicity can be kept low. This protocol describes the use of magnetic nanoparticles for delivery of nucleic acid to target cells, using either nonviral or viral vectors.

This protocol describes an approach to two-photon calcium imaging in dendrites and spines of living neurons. The technique can be applied to acute slices from hippocampus, cerebellum, and neocortex, as well as to slice and neuronal cultures. It uses two fluorescence detection channels to provide quantitative estimates of calcium concentration and to minimize the required concentrations of calcium indicator. A method for estimating calcium concentrations from fractional changes in fluorescent light intensity (F/F) is presented, along with two methods for loading neurons with calcium indicator.

This protocol describes an in vivo technique for combining whole-cell (WC) recordings of membrane potentials of individual neurons with voltage-sensitive dye (VSD) imaging of the ensemble neocortical network dynamics. If careful experimental measurements and controls are performed, VSD imaging can be used to define the ensemble spatiotemporal subthreshold membrane potential dynamics within which the membrane potential changes of individual neurons are embedded. Data from these individual neurons can be recorded simultaneously with the ensemble dynamics using the WC technique. This approach has been tested on rodent barrel cortex, but it is likely to be applicable, with minor modifications, to other superficial brain areas (including other neocortical areas, cerebellum, and olfactory bulb) and other species (such as cat or monkey).

The closed-loop-stripping technique with absorber traps (e.g., charcoal, porous polymers) can be used to collect volatiles from different biological sources, for example, plants and insects. One notable advantage of the method is the high signal-to-noise ratio that can be achieved by continuous sampling of the volatiles from a producing source within a closed environment. The equipment required for the technique is inexpensive and can be easily adapted to the demands of the analyzed subject. Moreover, the system can be operated in the field since only air and a battery are needed. The volatiles can be recovered from the trap by thermodesorption or by elution with solvents. Solvent elution has the advantage that unknown compounds can be subjected to chemical transformations facilitating structure elucidation. Only very low amounts of solvents (20-40 µL) are needed to elute the microcarbon traps (1.5-5 mg). Single traps efficiently absorb volatiles up to the lower microgram range. Higher concentrations may result in insufficient trapping. In this protocol, we describe the collection of volatiles released from lima bean leaves (Phaseolus lunatus) after damage by feeding larvae of Spodoptera littoralis. The enclosed volume of air containing the emitted volatiles is circulated through miniature charcoal traps for 4 h or more. For analysis, the traps are eluted with dichloromethane.

The crucial roles of RNA-binding proteins in all aspects of RNA metabolism, particularly in the regulation of mRNAs and subsequent control of gene expression patterns, have become increasingly evident. RNA immunoprecipitation (RIP) is a powerful technique used to detect the association of individual proteins with specific RNA molecules in vivo. Live cells are treated with formaldehyde to generate protein-RNA cross-links between proximal molecules. Following immunoprecipitation of a protein of interest and cross-link reversal, associated RNAs can be recovered, characterized, and quantitated by reverse transcriptase polymerase chain reaction (RT-PCR). Protein association with specific RNA regions can be performed under a variety of conditions (e.g., different environmental and cell-cycle states) and/or in mutant strains. Furthermore, because formaldehyde inactivates cellular enzymes essentially immediately upon addition to cells, RIP provides snapshots of protein-RNA interactions at specific time points and hence is useful for kinetic analyses of events occurring on RNA in vivo. The basics of RIP are very similar to chromatin immunoprecipitation (ChIP), but with some caveats that are important to appreciate to take full advantage of the possibilities afforded by RIP.

The use of zebrafish (Danio rerio) as a model organism has seen rapid growth in recent years. Numerous new techniques have emerged for zebrafish, and many protocols used in other model organisms have been adapted to fit the special needs of this species. Zebrafish cell lines, however, have not been studied to a great extent, and their applications remain limited. The PAC2 fibroblast line, isolated from 24-h post-fertilization zebrafish embryos, is one of the few available lines. Here, we provide a basic set of methods for maintenance and handling of PAC2 cells, as well as general procedures for transfection, immunocytochemistry, and protein extraction.

Systematic mutational analysis is required for the comprehensive deciphering of gene function. However, repeated targeting of a single locus is labor intensive and has not been a routine approach for studies using multicellular organisms. We have developed the "site-specific integrase mediated repeated targeting" (SIRT) method to facilitate targeted mutagenesis in Drosophila melanogaster. In SIRT, homologous recombination is used to place a landing site for the phage phiC31 integrase in the vicinity of the target locus. All subsequent genetic modifications to the same gene are introduced by integrase-mediated precise insertion of plasmids directly injected into embryos. For SIRT mutagenesis, one must generate a series of plasmid vectors that contain various DNA elements placed at different positions in the target-homologous clone. Unlike traditional cloning methods, SIRT is not limited by the availability of convenient restriction cut sites. This protocol presents the details of SIRT plasmid construction, relying heavily on the method of bacterial recombineering and using a number of streamlined DNA elements.

High-throughput whole-genome analysis has become a practical and important technique to understand nuclear processes, such as transcription, replication, and genome structure. Though microarrays have been the preferred genome-scale analysis method for over a decade, new technologies, referred to as next-generation sequencing, offer distinct advantages over microarrays in both sensitivity and scale. Several next-generation sequencing platforms are currently available, including the Genome Analyzer (Solexa/Illumina), 454 (Roche), and ABI-SOLiD (Applied Biosystems). This protocol describes sample preparation for sequencing of chromatin-immunoprecipitated DNA (ChIP-Seq) to analyze histone modification patterns using native chromatin and the Genome Analyzer. One advantage of using native chromatin as compared to cross-linked chromatin is that it provides single-nucleosome-level resolution and avoids nonspecific modification signals from different nucleosomes carried over through protein-protein interactions. The protocol includes purification of human CD4+ T cells from lymphocytes and chromatin fragmentation using micrococcal nuclease (MNase) digestion, followed by chromatin immunoprecipitation (ChIP) and construction of a library for sequencing.

Genetically modified adenoviruses (Ads) make attractive vectors for the delivery of exogenous DNA to mammalian cells for basic science and gene therapy applications. Ad vector production consists of (1) cloning a transgene into an infectious plasmid by in vivo recombination in bacteria, (2) rescuing and propagating the vector in complementing cells, and (3) purifying the vector. All of this can be accomplished using commercially available reagents, plasmids, and cell lines. First-generation Ads have a large cloning capacity (5-14 kbp) and efficiently transduce a wide range of both quiescent and proliferating cell types. They are readily propagated to produce high-titer stocks (10¹¹-10¹³ vector particles from a 3-L culture). Furthermore, Ads rarely integrate into the host genome and are relatively safe. However, Ad vector production typically takes 4-6 wk, and promiscuous host-cell transduction can occur in vivo. Furthermore, immune responses against viral proteins encoded by the vector backbone can occur, which limits the duration of transgene expression in vivo. Regardless of these limitations, Ad remains one of the more versatile and efficient gene delivery systems. Here, we discuss methods for the generation, propagation, purification, and characterization of first-generation Ad vectors.

Axis and germ-layer formations are central issues in vertebrate embryology. An intriguing question is how the mechanisms that existed in an ancestral vertebrate have been modified during vertebrate evolution. A major stream of vertebrates (Osteichthyes) evolved in two monophyletic lineages: the Sarcopterygii and Actinopterygii. There are many differences in the molecular and cellular mechanisms of embryogenesis between teleosts (actinopterygians) and amphibians (sarcopterygians), including the differences in bauplan (body plan). Polypterus diverged from all other actinopterygians ~400 million years ago (Mya) during the Devonian period, soon after the divarication of an ancestral bony fish into Actinopterygii and Sarcopterygii. Polypterus is thus uniquely well suited for studies assessing the ancestral state or bauplan of Osteichthyes and Actinopterygii, as well as the divergence of embryogenetic processes in teleosts and amphibians. This protocol describes the collection of Polypterus embryos and the method for microinjection.

Axis and germ-layer formations are central issues in vertebrate embryology. An intriguing question is how the mechanisms that existed in an ancestral vertebrate have been modified during vertebrate evolution. A major stream of vertebrates (Osteichthyes) evolved in two monophyletic lineages: the Sarcopterygii and Actinopterygii. There are many differences in the molecular and cellular mechanisms of embryogenesis between teleosts (actinopterygians) and amphibians (sarcopterygians), including the differences in bauplan (body plan). Polypterus diverged from all other actinopterygians ~400 million years ago (Mya) during the Devonian period, soon after the divarication of an ancestral bony fish into Actinopterygii and Sarcopterygii. Polypterus is thus uniquely well suited for studies assessing the ancestral state or bauplan of Osteichthyes and Actinopterygii, as well as the divergence of embryogenetic processes in teleosts and amphibians. This protocol describes a method for in situ hybridization in Polypterus embryos.

Computer prediction of the interaction between enzymes and small molecules has now advanced to the point that it allows accurate prediction of bound conformations and binding constants. For instance, the program AutoDock allows consistent computational docking of flexible ligands with about a dozen torsional degrees of freedom, and the empirical free-energy force field provides predicted energies that are accurate to within ~2 kcal/mol, or an ~30-fold difference in binding constants. Thus, these methods can easily separate compounds with micromolar and nanomolar binding constants from those with millimolar binding constants, and can often rank molecules with finer differences in affinity. Computational docking methods can be used to screen a variety of possible compounds, searching for new compounds with specific binding properties or testing a range of modifications of an existing compound. The approach has been successful in numerous cases, most notably, the discovery of human immunodeficiency virus (HIV) protease inhibitors. This protocol presents a detailed outline and advice for use of AutoDock and its graphical interface, AutoDock Tools, to analyze biomolecular complexes using computational docking. The first step is to prepare the coordinate files for the docking molecule and the target molecule. The second step is the calculation of the affinity grid for the target molecule. In the third step, the docking molecule is docked with the affinity grid, and, finally, the results are analyzed.

This protocol describes methods required for the culture of the African butterfly Bicyclus anynana. The larvae are typically fed and maintained on pot-grown maize plants (Zea mays) that are ~50 cm high. Males and females can be separated as pupae or adults. Adults are fed on moist banana and will readily mate in the laboratory. Females lay eggs on available grass plants. We use a standard temperature of 27°C (because an approximate rule is that developmental time is twice as long at 20°C), a high relative humidity of ~60%-70% (the exact level is not critical), and a 12:12 photoperiod similar to the climate experienced in the wet season near the equator. Both larval and pupal molts are gated by photoperiod and can be readily timed (e.g., by use of time-exposure filming). To set up cohorts of standard developmental stages, an appropriate timing for this photoperiod is chosen: Pupation usually occurs shortly after lights out, and larval molts also occur during the night. At ~27°C, egg development takes 4 d, and the total generation time is 5-6 wk, yielding about eight generations a year in selection experiments.

This protocol describes cautery experiments with young pupal wings of the African butterfly Bicyclus anynana to examine the potential role of groups of cells in wing pattern determination. Experiments can be performed on either the left or right wing, leaving the other wing as a control. The pupal wing case overlies a stack of four sheets of epithelial cells that will form, in turn, the dorsal and ventral surfaces of the forewing and then the respective surfaces of the hindwing. The dorsal surface of the forewing is immediately underneath the cuticle. The trachea or wing veins of the forewing are visible through the pupal cuticle under a binocular microscope, providing landmarks. Raised "bumps" or irregularities on the pupal cuticle indicate where the eyespot organizers (or foci) are present on the underlying forewing. The wild-type pattern of the dorsal forewing consists of a small anterior eyespot and a large posterior eyespot; each eyespot has a central white pupil, a black inner ring, and an outer gold ring. Experiments that involve a short insertion of sharpened needles through the pupal cuticle can explore the effects of damage to groups of cells at different times from shortly after pupation until pattern determination has occurred (~24 h after pupation). This type of experiment originally helped to characterize the eyespot organizers because early focal damage to the location of these cells can result in shrinkage or complete elimination of the corresponding eyespot in the adult wing (relative to the control wing).

This protocol describes a procedure for transplanting tissue of the eyespot organizer in early pupae of the African butterfly Bicyclus anynana. In very young pupae, before apolysis, the pupal wing case is attached to the sheet of cells that will form the dorsal surface of the adult forewing. Therefore, if a piece of pupal cuticle is removed and transplanted to a novel site on the developing wing (where a corresponding patch has been removed), the underlying cells of the dorsal forewing are transferred together with it. The pattern determination effects of the transplanted wing tissue on the surrounding cells can be examined by comparing the experimental and control wings of the adult butterfly. Square grafts can be turned 180° (or 90°) before implantation to distinguish donor and host tissue, because the orientation of the scale cells is determined before grafting.

This protocol describes a procedure for the fixation and dissection of embryos from the African butterfly Bicyclus anynana. Such embryos can be used subsequently for in situ hybridization experiments or for immunohistochemistry analysis.

This protocol describes a procedure for the dissection of larval and pupal wings from the African butterfly Bicyclus anynana. Dissected pupal wings can be used subsequently for in situ hybridization or for immunohistochemistry. They can also be analyzed with stereomicroscopy to study scale maturation and pigment deposition.

The modified in situ hybridization protocol described here can be used to localize RNA transcripts in developing tissues of the African butterfly Bicyclus anynana, including larval and pupal wings and embryos.

This protocol describes an immunohistochemical method for the detection of proteins in developing embryos of the African butterfly Bicyclus anynana. The embryos are stained with a primary antibody against a protein of interest, followed by a fluorescently labeled secondary antibody.

This protocol describes an immunohistochemical method for the detection of proteins in developing wings of the African butterfly Bicyclus anynana. The wing discs are stained with a primary antibody against a protein of interest, followed by a fluorescently labeled secondary antibody.

Pheromones are used for communication among individuals of the same species, including attracting mates. A blend of chemical components is recognized as a pheromone if it elicits a behavioral or physiological response. Gas chromatography (GC) helps to separate complex mixtures of chemicals extracted from a target tissue. When the extract is associated with an internal standard, GC can be used to quantify the amounts of the chemical components present on a target tissue. This protocol describes the preparation of pheromone extracts from wings of the African butterfly Bicyclus anynana and the subsequent use of these extracts in GC analysis.

For many biological processes, such as metabolic rate, starvation resistance, and fecundity, knowledge of body composition is important. This protocol describes how to measure fresh weight (FW), dry weight (DW), and fat-free dry weight (FFDW) of African butterfly Bicyclus anynana adults. From this, the weight of water and fat can be calculated, as well as water and fat fractions.

This protocol describes the use of constant volume respirometry to measure CO₂ production by the African butterfly Bicyclus anynana. The instrument used is the TR-2 system from Sable Systems International. In this setup, dry CO₂-free air is sequentially pumped through 16 sealed chambers. The CO₂ produced by the butterflies is subsequently measured with an infrared CO₂ detector. CO₂ production (µL/ h) by individual butterflies is an index of the resting metabolic rate (RMR). The RMR of each individual is corrected for weight, preferably after removal of water and fat mass, to allow comparison of the RMRs among individuals.

This protocol describes procedures for the extraction of hemolymph from larvae, pupae, and adults of the African butterfly Bicyclus anynana. The hemolymph can subsequently be used for various purposes; the method for processing the hemolymph depends on the question under investigation.

This protocol describes a procedure for the injection of chemicals into pupae of the African butterfly Bicyclus anynana. A number of target traits can be monitored to determine the effect of a particular chemical on the pupae.

The use of antibodies to visualize the distribution and subcellular localization of gene products powerfully complements genetic and molecular analysis of gene function in Caenorhabditis elegans. The challenge to immunolabeling C. elegans is finding the fixation and permeabilization methods that effectively make antigens accessible without destroying the tissue morphology or the antigen. Embryos are surrounded by a chitinous eggshell and larvae and adults are surrounded by a collagenous cuticle, each of which must be permeabilized to allow penetration of antibodies. In addition, antigens and antibodies are sensitive to different fixing and permeabilizing conditions. This protocol describes two methods for tissue fixation. The whole-mount freeze-cracking method is a good starting point as it is easy and works well with most antibodies and with embryos, larvae, and adults. In the tissue extrusion method, gonads and intestines, which are extruded from the carcass, are well fixed and permeabilized. Tissues remaining in the carcass are not usually stained well. The protocol concludes with an antibody incubation procedure in which fixed worms are incubated overnight with primary antibody, subsequently exposed to secondary antibody, and mounted for viewing.

Therapeutic gene transfer strategies aim to maximize gene transfer and expression in target cells. However, gene delivery systems often fail to preferentially transduce target cells in mixed cell populations. In addition, limitations in vector specificity can lead to transduction of non-target cells, resulting in untoward toxicity, even with compartmental dosing. Thus, vector optimization is critical for the development of efficient genetic experiments. The life cycle and biology of adenovirus (Ad) infection and transgene expression in cells have been thoroughly characterized. Ad infection is initiated by recognition of the native Ad5 receptor, coxsackievirus-adenovirus receptor (CAR), on target cells by the carboxy-terminal portion (i.e., knob) of the fiber protein. The development of genetically modified Ad vectors with transductional specificity for a single cell type requires "retargeting": the ablation of endogenous tropism and the introduction of novel tropism determinants for target cells. This protocol describes the construction of chimeric fibers. Homologous recombination between the pAdEasy-1 and the fiber shuttle plasmid (pFiber-dE3-RGD) is used to generate a rescue plasmid with a mutant fiber that inserts Arg-Gly-Asp (RGD) at the carboxyl terminus of the fiber knob region. The resultant fiber-modified rescue plasmid (pAdEasy-RGD) is isogenic to AdEasy-1 (except the fiber region) and can therefore be used for another round of homologous recombination with pShuttle-promoter-Luc to make a final Ad vector construct that expresses the luciferase gene and contains an RGD-modified fiber knob. This construct can then be tested for targeting efficacy against cells expressing _v integrin cell surface receptors.

Therapeutic gene transfer strategies aim to maximize gene transfer and expression in target cells. However, gene delivery systems often fail to preferentially transduce target cells in mixed cell populations. In addition, limitations in vector specificity can lead to transduction of nontarget cells, resulting in untoward toxicity, even with compartmental dosing. Thus, vector optimization is critical for the development of efficient genetic experiments. The life cycle and biology of adenovirus (Ad) infection and transgene expression in cells have been thoroughly characterized. Ad infection is initiated by recognition of the native Ad5 receptor, coxsackievirus-adenovirus receptor (CAR), on target cells by the carboxy-terminal portion (i.e., knob) of the fiber protein. The development of genetically modified Ad vectors with transductional specificity for a single cell type requires "retargeting:" the ablation of endogenous tropism and the introduction of novel tropism determinants for target cells. One strategy for accomplishing such retargeting does so indirectly, by using bifunctional adapter molecules. One element of the bispecific adapter binds to the Ad fiber knob, blocking its interaction with CAR and, thus, its native tropism. The second component of the adapter introduces specificity for the target cells and is chemically or genetically conjugated to the knob-binding portion. Recombinant fusion proteins offer a number of technological advantages over chemical conjugates, including simplified production and purification. In this protocol, a recombinant fusion protein is generated that ablates the vector’s native tropism by blocking the CAR receptor and retargets the vector to cells expressing epidermal growth factor (EGF).

The balance between target and nontarget cell toxicity determines the efficacy of any therapeutic agent. Therapeutic gene transfer strategies aim to maximize gene transfer and expression in target cells. However, gene delivery systems often fail to preferentially transduce target cells in mixed cell populations. In addition, limitations in vector specificity can lead to transduction of nontarget cells, resulting in untoward toxicity, even with compartmental dosing. Thus, vector optimization is critical for the development of efficient genetic experiments. The life cycle and biology of adenovirus (Ad) infection and transgene expression in cells have been thoroughly characterized. While genetically modified Ad vectors with transductional specificity for a single cell type can be generated by direct or indirect modifications of the carboxyl terminus of the vectors’ receptor recognition site, Ad vectors can also be targeted at the level of protein expression of the transgene in the targeted cells (i.e., transcription/translation). Target-cell-specific gene expression is accomplished using tissue-specific promoters (TSPs), DNA elements that restrict expression to specific cellular subsets. The major drawback of transcriptional retargeting is that pathological change in target tissues or organs, such as degeneration or malignant transformation, can ectopically activate TSPs. In this protocol, recombinant Ad vectors that express the luciferase gene are constructed through homologous recombination in Escherichia coli. TSPs are placed in front of the luciferase gene for selective expression.

The Comet-FISH technique is a useful tool for detection of overall and region-specific DNA damage and repair in individual cells. It combines two well-established methods, the Comet assay (single-cell gel electrophoresis) and fluorescence in situ hybridization (FISH). The Comet assay serves as the basis of Comet-FISH and is a relatively simple and fast method that allows separation of fragmented from nonfragmented DNA and quantification of DNA damage and repair. FISH enables detection of specifically labeled DNA sequences of interest, including whole chromosomes. The combined technique of Comet-FISH is a modification of the Comet assay that inserts a hybridization step after unwinding and electrophoresis and permits the labeling of specific gene sequences or telomeres. Comet-FISH has been applied for detection of site-specific breaks in DNA regions that are relevant for development of various diseases, and has also been used to study the distribution of DNA damage and repair in the complete genome. Moreover, DNA sequence modifications can be detected in individual cells using Comet-FISH. Whereas results from the Comet assay alone reflect only the level of overall DNA damage, the addition of the FISH technique allows the assignment of the probed sequences to the damaged or undamaged part of the comet (tail or head, respectively).

Many polypeptides do not perform their functions as single autonomous units in vivo. Instead, multiple polypeptides associate to form higher molecular mass structures. Blue-native polyacrylamide gel electrophoresis (BN-PAGE) allows a range of the major protein complexes involved in such protein-protein interactions to be visualized simultaneously and in a single experiment. When combined with a second dimension of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the BN/SDS-PAGE procedure can resolve the complexes according to their molecular weight, as well as the subunits within each complex, according to the molecular weights of the subunits. Similarly, used in conjunction with differential in-gel electrophoresis (DIGE), it can accurately quantify changes in protein complex abundance or subunit composition between different samples, or between different complexes within the same sample. The following basic protocol describes sample preparation and gel casting for the first (BN-PAGE) and second (SDS-PAGE) dimensions. Variants are presented with and without DIGE labeling, along with the additional steps required for the fluorescence DIGE technique.

Mass spectrometric analysis of the metabolome (the complete set of small-molecule metabolites such as metabolic intermediates, hormones, and other signaling molecules, and secondary metabolites to be found within a biological sample, such as a single organism) has become a key component of modern systems biology. Within this broad definition, a number of technical refinements have enabled high-throughput profiling to be performed of the exometabolome (the metabolites that a cell or system excretes under controlled conditions). This technique has become known as metabolic footprinting and was first developed for analysis of yeast single- gene deletion mutants. More recently, it has proved valuable in metabolic profiling and comparative analysis of brewing and medically important yeast biodiversity. Direct injection mass spectrometry (DIMS) enables the direct injection or infusion of a sample, typically into an electrospray ionization mass spectrometer (ESI-MS). This article describes a detailed DIMS methodology in which samples of spent media from yeast cultures are introduced into an ESI-MS by direct injection into a flowing solvent (flow injection). DIMS resolves complex mixtures into components differing in ion mass using electrospray ionization, which avoids the need for derivatization and time-consuming chromatographic separation that is found with gas chromatography-mass spectrometry (GC-MS). The method is rapid and discriminatory. Populations of extremely closely related yeast strains can be identified and assigned to clusters of comparable phenotypes, even when standard genetic fingerprinting techniques fail to discriminate among such variants.

The goal of chromatin assembly procedures is to prepare extended nucleosomal arrays from cloned DNA templates and purified core and linker histones. The assembled chromatin should be highly defined in its protein content and resemble bulk chromatin isolated from living cell nuclei in terms of periodicity and nucleosome positioning. This protocol describes how to assemble minichromosome templates in an ATP-dependent fashion from circular plasmid DNA and purified core histones. This system can also be used to assemble minichromosomes from linear DNA (plasmid and ) and can also incorporate proteins other than core histones (linker histone H1, HMG17, and DNA-binding transcription factors). The products of the chromatin assembly reaction can be used directly (or after purification) in assays to study transcription, DNA replication, recombination, and repair. The system uses purified recombinant Drosophila chromatin assembly factors ACF (ATP-utilizing chromatin assembly and remodeling factor) and NAP1 (nucleosome assembly protein 1).

The goal of chromatin assembly procedures is to prepare extended nucleosomal arrays from cloned DNA templates and purified core and linker histones. The assembled chromatin should be highly defined in its protein content and resemble bulk chromatin isolated from living cell nuclei in terms of periodicity and nucleosome positioning. This protocol describes the preparation of Drosophila ACF (ATP-utilizing chromatin assembly and remodeling factor) for use in chromatin assembly reactions. In this method, ACF is prepared by the coexpression of the carboxyl-terminally FLAG-tagged Acf1 subunit with the untagged ISWI subunit in baculovirus. The complex is then purified in one step by FLAG immunoaffinity chromatography. This procedure typically results in a stoichiometric complex of Acf1 and ISWI.

The goal of chromatin assembly procedures is to prepare extended nucleosomal arrays from cloned DNA templates and purified core and linker histones. The assembled chromatin should be highly defined in its protein content and resemble bulk chromatin isolated from living cell nuclei in terms of periodicity and nucleosome positioning. This protocol describes the preparation of the NAP1 chaperone protein for use in chromatin assembly reactions. In this method, Sf9 cells are infected with an HIS6-DNA-1-expressing baculovirus. The NAP1 is then purified by nickel affinity chromatography, followed by anion-exchange chromatography.

The marine gastropod Ilyanassa obsoleta is a long-standing and very useful model for studies of embryonic development. It is an especially important model for spiralian development, and for studies of asymmetric cell division. The embryos are amenable to classic embryological manipulation techniques as well as a growing number of molecular approaches. Ilyanassa is also an important model for studies of metamorphosis, the ecology of parasitism, the effects of environmental contaminants on morphology and sexual function, and comparative neurobiology. Ilyanassa adults are readily obtainable and easy to keep in the laboratory. Although the normal spawning season for Ilyanassa is during early summer, they can produce high-quality embryos nearly year-round in the laboratory. Snails collected in the late fall, winter, or spring can be induced to deposit zygotes before the natural spawning season by warming them to room temperature, and snails collected before the natural spawning season can be made to postpone zygote deposition until needed (up to at least 6 mo) by maintaining them in tanks in a cold room at 4°C-8°C. This protocol describes how to induce embryo production in Ilyanassa snails, collect the embryos, and rear them to the stage required for study.

The marine gastropod Ilyanassa obsoleta is a long-standing and very useful model for studies of embryonic development. It is an especially important model for spiralian development, and for studies of asymmetric cell division. The embryos are amenable to classic embryological manipulation techniques as well as a growing number of molecular approaches. Ilyanassa is also an important model for studies of metamorphosis, the ecology of parasitism, the effects of environmental contaminants on morphology and sexual function, and comparative neurobiology. Ilyanassa adults are readily obtainable and easy to keep in the laboratory, and they can produce high-quality embryos nearly year-round in the laboratory. After hatching from capsules, larval Ilyanassa can be maintained in culture, feeding on single-celled algae. The larvae will become competent to undergo metamorphosis after ~3 wk in culture. Metamorphosis can be induced artificially by treating with either the neurotransmitter serotonin or the nitric oxide synthase inhibitor 7-nitroindazole. Both of these reagents have been shown to induce metamorphosis in >75% of larvae within 48 h. This protocol describes the induction of metamorphosis in snail larvae.

The marine gastropod Ilyanassa obsoleta is a long-standing and very useful model for studies of embryonic development. It is an especially important model for spiralian development, and for studies of asymmetric cell division. The embryos are amenable to classic embryological manipulation techniques, as well as a growing number of molecular approaches. Ilyanassa is also an important model for studies of metamorphosis, the ecology of parasitism, the effects of environmental contaminants on morphology and sexual function, and comparative neurobiology. Intracellular microinjection is an important tool, especially for lineage tracing and perturbations of specific genes by knockdown approaches and synthetic mRNA injections. Two methods for the introduction of lineage tracers into particular cells are routine in Ilyanassa. Iontophoresis of charged molecules, such as fluorophore-dextran conjugates can be accomplished using a simply built current generator. Injection of an oil-based solution containing the fluorescent probe 1,1-dioctadecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate (DiI) is also straightforward. However, injection of oil-based solutions and iontophoresis have not been useful for delivering water-soluble reagents to perturb gene function, and pressure injection of aqueous solutions has been more challenging. This protocol describes a recently optimized procedure for the pressure injection of aqueous solutions into Ilyanassa embryos and zygotes with high rates of survival and normal development. The key parameters seem to be the injection needles, injection media, and the stage of injected embryos.

The marine gastropod Ilyanassa obsoleta is a long-standing and very useful model for studies of embryonic development. It is an especially important model for spiralian development, and for studies of asymmetric cell division. The embryos are amenable to classic embryological manipulation techniques, as well as a growing number of molecular approaches. Ilyanassa is also an important model for studies of metamorphosis, the ecology of parasitism, the effects of environmental contaminants on morphology and sexual function, and comparative neurobiology. Ilyanassa embryos are particularly well suited for RNA and protein localization studies because of the relatively large cells and favorable properties for imaging. This protocol describes how to fix and store Ilyanassa embryos and larvae for use in whole-mount in situ hybridization and immunohistochemical studies.

The marine gastropod Ilyanassa obsoleta is a long-standing and very useful model for studies of embryonic development. It is an especially important model for spiralian development, and for studies of asymmetric cell division. The embryos are amenable to classic embryological manipulation techniques as well as a growing number of molecular approaches. Ilyanassa is also an important model for studies of metamorphosis, the ecology of parasitism, the effects of environmental contaminants on morphology and sexual function, and comparative neurobiology. Ilyanassa is host to several species of parasitic trematode worms, so care must be taken to avoid contamination of Ilyanassa genomic DNA with that of the parasites. The easiest way to avoid this contamination is to isolate DNA from veliger larvae, which are not parasitized. This also avoids other problems that can be encountered when isolating DNA from adult mollusc tissues, such as the presence of large amounts of polysaccharides. This protocol describes the isolation of genomic DNA from Ilyanassa larvae.

The marine gastropod Ilyanassa obsoleta is a long-standing and very useful model for studies of embryonic development. It is an especially important model for spiralian development, and for studies of asymmetric cell division. The embryos are amenable to classic embryological manipulation techniques as well as a growing number of molecular approaches. Ilyanassa is also an important model for studies of metamorphosis, the ecology of parasitism, the effects of environmental contaminants on morphology and sexual function, and comparative neurobiology. This protocol describes the procedure for extracting protein from Ilyanassa embryos for use in techniques such as Western blotting or two-dimensional (2D) gel electrophoresis.

Gravid Helobdella (leech) adults are identified by white egg masses that are visible through the ventral body wall. For convenience, gravid animals should be moved to smaller bowls so that those animals with newly laid embryos can be more readily identified. Zygotes (fertilized internally) turn pink when they are deposited into transparent cocoons on the ventral surface of the parent. A typical clutch consists of 20-100 embryos. Embryos can be collected after zygote deposition and before hatching (stage 10). This article describes how to collect and culture Helobdella embryos.

One advantage of using Helobdella (leech) embryos as an experimental system is their amenability for microinjection. Blastomeres ranging in size from the zygote (400 µm diameter) down to micromeres and primary blast cells (~20 µm diameter) can be injected by pressure under a dissecting microscope. Smaller cells can be injected by iontophoresis under a compound microscope. Microinjection is useful for studying embryonic development. For example, developmental fates of a cell can be followed by injecting a lineage tracer. A specific cell can be killed by injecting a toxic substance. Furthermore, cells can be killed at a given developmental stage by directing intense fluorescence illumination or a blue laser beam on fluorescein-labeled cells. Finally, gene expression can be manipulated in leech embryos by injecting zygotes or selected blastomeres with synthetic mRNA, morpholino antisense oligo, or a plasmid construct. Molecules <1500 Da can diffuse freely among early blastomeres via gap junctions. When intercellular diffusion of an injected substance is undesirable, small molecules should be conjugated to larger molecules such as dextran (10 kDa). Different commercial microinjection setups can be adopted for Helobdella embryos. This article describes how to microinject embryos using a versatile homemade pressure injection system. Under a dissecting microscope, embryos are immobilized by suction in a custom-fabricated chamber with the target cell facing upward. Cells are visualized using transillumination via a long-working-distance, dark-field condenser. The tip of a micropipette is brought into the target cell with a micromanipulator, and the injectant is delivered into the cell by pressure.

Embryos of glossiphoniid leeches are enclosed in a thin vitelline envelope until "hatching" (stage 10). This protocol describes the procedure for removing the vitelline envelope from Helobdella robusta and H. sp. (Austin) embryos. This protocol is applicable to embryos of stages 1-9 but is probably more useful for early stages. With careful culturing, the devitellinized embryos can develop normally.

Arnolds’ silver staining is used to visualize the boundaries of epidermal cells at the surface of embryos. As described in this article, silver staining can be performed on either living or fixed Helbodella (leech) embryos. To preserve the cell junction in fixed embryos, a special fixation procedure is used.

The purpose of this staining protocol is to detect the localization of specific antigens in the Helobdella (leech) embryo. Immunostaining protocols must be optimized for each antibody and embryonic stage. Here, as a starting point, we present a general-purpose immunostaining protocol. Immunostaining of the leech embryo can be performed on the intact embryo or on dissected parts. For post-gastrulation embryos (stages 9 and 10), if the area of interest is in the germinal plate, immunostaining can be performed on the dissected germinal plate. It can also be performed on the ventral nerve cords dissected from late-stage embryos or juveniles.

The purpose of this protocol is to reveal the localization of transcripts in the Helobdella (leech) embryo. In situ hybridization protocols for Helobdella embryos are derived from those used for zebrafish and Xenopus. The protocols are different for early- and late-stage embryos (stages 1-8 and stages 9-11, respectively) due to a difference in tissue permeability.

Due to the high yolk content and relatively large size of the Helobdella (leech) embryo, it is preferable to examine the embryos in a cleared whole-mount preparation after they have been stained. Three whole-mount procedures suitable for leech embryos are described here. The glycerol whole mount is quick and convenient because it does not require dehydration. However, prolonged incubation in buffered glycerol could cause the yolk to turn dark purple. Thus, it is not suitable for yolk-containing specimens that are intended for light microscopy. For fluorescence microscopy, anti-fading reagent can be added to the buffered glycerol. This article includes a recipe that works quite well for leech embryos. Also included are two alternative methods that require embryo dehydration, but have the advantage of clearing yolk-containing tissue. One method relies on the use of benzyl benzoate:benzyl alcohol (BBBA), a solution that provides the best clearing of yolk-containing tissue in the leech embryo. It is suitable for both fluorescence and light microscopy. Another alternative is the Epon whole-mount preparation. Epon is not quite as effective in clearing the yolk, but the viscosity of Epon facilitates orienting the specimens for imaging. As with BBBA, Epon is suitable for both light and fluorescence microscopy.

Transfer RNA intergenic spacer length polymorphism analysis (tDNA-PCR) is a simple and reproducible polymerase chain reaction (PCR) technique for identification of bacteria at the species or even subspecies level. The primers used in the PCR are based on conserved sequences located at the edges of the tRNA genes. Because the selected consensus primers are directed outwardly, the intergenic spacers are amplified rather than the genes themselves. With each PCR, several amplicons of different lengths are obtained, because several intergenic spacers are present in each bacterial genome. The patterns thus obtained are identical within species, but differ between distinct species, and as a result, can be used for identification of bacterial species. The amplicons are separated using high-resolution (1 bp) electrophoresis (e.g., fluorescent capillary electrophoresis) and immediately digitized as tables composed of numerical lengths (expressed in base pairs) and peak intensities. For identification, the resulting peak pattern can be compared with a large database of patterns of well-identified bacterial strains, using an in-house-developed software package that is available online. New patterns (linked to the correct species name, which can be obtained, e.g., after 16S rRNA gene sequence determination) can be added to expand the database further. This protocol describes tDNA-PCR, followed by automated fluorescent capillary electrophoresis to identify bacterial species.

Structural analyses of large protein-DNA complexes (such as those associated with replication initiation, eukaryotic transcription activation or chromatin remodeling, among others) remain a challenge because of difficulties in obtaining multi-milligram quantities of high-quality preparations of large, linear DNA molecules. This protocol describes a three-stage DNA amplification procedure for making such molecules in amounts that are suitable for structural studies. In the first step, conventional polymerase chain reaction (PCR) using specialized primer sequences is used to prepare a DNA molecule suitable for self-primed DNA synthesis. This molecule, which consists of the sequence of interest flanked by the cohesive end sequences from bacteriophage as well as endonuclease recognition sites, is then submitted to self-primed DNA synthesis in the second step. Amplification produces long polymers of DNA, tens of kilobases in length, which harbor many copies of the sequence of interest. The yield from the second step is increased in the third phase, which consists of another round of amplification. Finally, endonuclease digestion of these polymers, followed by chromatographic purification, yields high-quality preparations of DNA. The molecules produced by this procedure consist of the DNA sequence of interest with three base pairs at either end. This procedure typically yields 400-800 µg of purified DNA per milliliter of amplification reaction.

This protocol describes a standard miniprep for Drosophila melanogaster that requires very few flies and produces high-quality DNA. This method can also be used to isolate RNA when RNase-free conditions are utilized; an extra step must be taken to rid the sample of genomic DNA (e.g., RNase-free DNase digestion).

The Drosophila melanogaster P-transposable element is a powerful and widely used research tool. Sequences flanking the P-element can be recovered and the site of insertion can be mapped to the nucleotide, to connect the genetic and physical maps and facilitate molecular analysis of the gene of interest. The Berkeley Drosophila Genome Project (BDGP) has assembled a well-characterized collection of lethal mutations induced by single P-element insertions generated by a number of laboratories. The genomic DNA sequences adjacent to these insertions have been recovered by either plasmid rescue or inverse polymerase chain reaction (PCR). The combination of a complete genomic DNA sequence and relatively fast and easy molecular methods for mapping P-element insertion sites to the nucleotide enhances the use of P-elements as tools in Drosophila research. This protocol provides detailed procedures for isolating DNA flanking P-element insertions.

This protocol describes the loading of individual cells with fluorescent probes via patch pipettes. The patch-clamp methodology has been successfully used for single-cell dye labeling in cultured neurons, brain slices, and in vivo preparations. A broad range of dyes can be used with this loading technique. Markers for morphological reconstruction (e.g., Lucifer yellow); ion-sensitive indicator dyes for monitoring second-messenger cascades (e.g., fura-2); and dye-labeled proteins for fluorescence resonance energy transfer (FRET), fluorescence correlation spectroscopy (FCS), and fluorescence recovery after photobleaching (FRAP) studies are all suitable for patch-clamp loading. The most widespread application of this technique has been for Ca²⁺ imaging. Whole-cell patch-clamp recordings represent a versatile loading technique that allows combined electrophysiological and optical measurements at a quantitative level.

This protocol describes detailed procedures for rapid labeling of cells in a variety of preparations by means of particle-mediated ballistic (i.e., Gene Gun) delivery of fluorescent dyes. The method has been used for rapid labeling of cells with either lipid- or water-soluble dyes, in a variety of preparations at different ages. Tissue preparations include fixed mouse brain slices (described here), cell cultures, and tissue explants. This ballistic labeling technique is useful for studying neuronal connectivity, function, and pathology in the nervous system of living as well as fixed specimens.