Cold Spring Harbor protocols最新文献

Amino acid analysis is a vital part of analytical biochemistry. The increasing demand for low nitrogen fertilization and for plant-based diets with balanced amino acid levels and composition have made it crucial to develop reliable, fast, and affordable methods for analyzing amino acids in plants. As maize accounts for 43% of global cereal production, improving the amino acid composition of its kernels (i.e., seeds) is critically important for meeting the dietary requirements of humans and livestock. Moreover, amino acid quantification in maize leaves is necessary for improving yield prediction, stress sensing, and nitrogen use efficiency. Many amino acid quantification methods use reverse-phase high-pressure liquid chromatography and gas chromatography approaches to assess the amino acid content of maize tissues. Historically, these techniques involved the use of chemical derivatization, a chemical reaction that alters the properties of a compound to make it detectable or more sensitive to detection. Although accurate, these methods are time-consuming, expensive, and unsuitable for large populations. Here, we introduce two high-throughput methods for quantifying amino acids from large maize populations, such as those used for quantitative trait locus mapping, genome-wide association studies, and large mutant populations. Both methods use an ultraperformance liquid chromatography-tandem mass spectrometry instrument to quantify all 20 proteogenic amino acids in a maize tissue in a short run time. A dependable, affordable, and high-throughput method for quantifying amino acids in maize has important implications for assessing kernel quality, yield, and management efficacy, such as fertilizer usage and watering.

Larvae of the fruit fly Drosophila melanogaster are a popular and tractable model system for studying the development and function of sensorimotor circuits, thanks to the relative numerical simplicity of their nervous system and the wealth of available genetic tools to manipulate the anatomy, activity, and function of specific cell types. Researchers studying the role of a particular gene or cell type in sensorimotor circuit activity or function may wish to observe the effects of an experimental manipulation during behavior in the intact animal. Observing these effects, which may include changes in the intracellular calcium concentration or movement of small numbers of neurons, muscles, etc., typically requires high-spatial-resolution imaging, which poses several difficulties in the freely crawling larva. Freely crawling larvae can move quickly and with changeable heading, making manual or automatic tracking challenging; additionally, they may make three-dimensional movements, such as rearing, that can degrade imaging focus. These challenges are potentially solvable using advanced imaging and algorithmic tracking setups, but cost, space, or development time may be prohibitive. This protocol describes a simple and cost-effective method for placing larvae inside agarose channels, thereby restricting larval crawling to a single dimension and enabling higher-magnification time-series imaging of fluorescently labeled structures during many cycles of locomotion. By using larvae that express fluorescent calcium indicators in cells of interest, researchers can apply this method to study the effects of experimental manipulations on neural or muscular activity during behavior in the intact animal.

In the Drosophila nerve cord, much is known about the generation of neurons from neuronal stem cells. Over the lifetime of a neuron, the cumulative expression of genes within that neuron determines its fate. Furthermore, gene expression in mature neurons determines their functional characteristics. It is therefore useful to visualize neural gene expression, which is often done via staining with antibodies to a protein of interest. In cases where there is no antibody to a desired gene product, or when it is useful to detect RNA rather than protein products, fluorescent in situ hybridization chain reaction for RNA (HCR RNA-FISH, or HCR for this protocol) can be used to detect and quantify RNA expression. RNA molecules reside predominantly in the cell soma, so HCR can facilitate determining neuron identity because somata position within the nerve cord is stereotyped across animals. HCR provides high-amplitude, high-fidelity signals. In principle, HCR can be broken down into a detection/hybridization stage and an amplification stage. During detection/hybridization, a probe set hybridizes to multiple sequences within a target gene. In the amplification step, concatemerized fluorescent hairpins bind to the hybridized probes. This two-step process increases the specificity of the fluorescent signal and helps reduce the likelihood of background fluorescence compared to traditional in situ hybridization techniques where the hybridizing probe itself contains the fluorescent signal. Here, we describe a protocol for using HCR to study gene expression in the Drosophila embryonic and larval nerve cord. We also describe how to combine HCR with immunofluorescence staining.

In animals, movement is generated by the activity of motor circuits housed in the vertebrate spinal cord or the arthropod nerve cord. How motor circuits form is a fundamental question, with wide-ranging impacts on the fields of development, neurobiology, medicine, evolution, and beyond. Until recently, studying circuit assembly had been experimentally difficult, with a paucity of suitable models. Due to the introduction of novel neuroscience tools (calcium imaging, optogenetics, connectomics), Drosophila embryos and larvae can be used as models to study motor circuit assembly. Here, we briefly review the knowledge relevant to motor circuit assembly in Drosophila larvae. We discuss the larval body and its movements, larval neurons and circuits in the motor system, and how the generation of neural diversity starting from stem cells relates to circuit formation. The long-term goal of Drosophila research in this field is to identify developmental rules, determine when the rules apply, generate an integrated understanding of motor circuit development, and uncover molecular mechanisms driving the assembly process. Motor circuits are an ancient part of the nervous system, and so far, the developmental programs guiding motor circuit assembly appear to be largely conserved across phyla. Thus, as methods improve in other systems, findings in Drosophila will provide foundational concepts that will inspire hypotheses in those systems.

The Drosophila larval nerve cord, which is the equivalent of the vertebrate spinal cord, houses the circuits required to process somatosensory stimuli (e.g., tactile, temperature, vibration, and self-movement) and generate the patterned muscle contractions underlying movement and behavior. Within this complex structure reside many cell types and cellular processes, making it difficult to experimentally access, when compared to peripheral parts of the nervous system (i.e., primary sensory neuron dendrites, motor neuron axons and synapses, and muscles). Additionally, the neurons in the larval nerve cord have small cell bodies, precluding traditional electrophysiological approaches. As such, the function of neurons in the nerve cord is less well studied than other parts of the nervous system, severely limiting our understanding of how larvae process sensory information and generate movement. Ca²⁺-sensitive fluorescent proteins enable the study of neuronal activity in live, genetically tractable animals, even those with small neuronal cell bodies. In addition, live imaging of neurons within the nerve cord in whole, intact animals is possible because larvae are translucent, and the use of intact animals allows for the peripheral sensory neuron circuits to remain intact. Ca²⁺-sensitive fluorescent proteins increase their fluorescence when voltage-gated Ca²⁺ channels are opened in depolarized neurons. Here, we describe an assay where a Ca²⁺-sensitive fluorescent protein (GCaMP6m) is expressed under the control of a GAL4 driver in a subset of neurons that reside in a circuit for vibration sensation. External vibration (sound) stimulates sensory neurons that activate the cells expressing the Ca²⁺-sensitive fluorescent protein. Visualization of the calcium-induced fluorescent signal with microscopy allows for quantification of neuronal activity.

Neurons exhibit some of the most striking examples of morphological diversity of any cell type. Thus, when studying neurons, the morphology of each neuron must be considered individually. However, neurons densely populate the central nervous system (CNS), making it difficult to ascertain fine morphological features due to a lack of spatial resolution. In Drosophila, this problem can be partially resolved by using driver lines that express the yeast transcription factor GAL4 in subsets of neurons. GAL4 can activate the expression of other introduced genetic elements such as genes for fluorescent proteins or other markers under the control of the GAL4 upstream activation sequences (UAS effectors). However, even highly specific GAL4 lines often label sets of potentially morphologically heterogeneous neurons. Here, we describe a protocol for using the multicolor flip-out (MCFO) technique in Drosophila melanogaster to stochastically label individual neurons within a GAL4 expression pattern. MCFO relies on the binary GAL4/UAS expression system in Drosophila but adds additional control for how densely the neurons within a GAL4 expression pattern are labeled via user-controlled heat shock. Specifically, three discrete UAS effector elements containing the sequences for unique epitope tags (FLAG, HA, and V5) linked to a gene for nonfluorescent GFP can be independently expressed under the control of GAL4 only when a transcriptional stop sequence in the UAS promoter sequence has been removed by heat shock-induced recombination. This effectively labels multiple individual neurons with either one or a combination of epitope tags that can be spectrally resolved with immunofluorescence. The MCFO technique is ideal for researchers who want to determine morphological features of CNS neurons in wild-type or mutant backgrounds.

The UniformMu National Public Resource is a widely used, functional genomics tool for maize, constructed by backcross introgression of active Robertson's Mutator (Mu) transposons into the W22 inbred line, creating a large, searchable collection of lines that together carry transposon insertions in thousands of maize genes. This resource provides (1) a ready supply of freely available mutant seed stocks, each linked to mapped gene sequences; (2) uniform controls in an inbred background, for precision analysis of mutant phenotypes; (3) a reliable source of heritable mutants that are consistently recovered in stated lines; and (4) stable mutant lines with no Mu activity. This low-cost resource provides a consummate experimental system for linking gene sequences with their function in a species that has long served humanity, not only as a preeminent genetic model, but also as one of the world's most productive grain crops. Here, we describe how to perform an initial, online search of insertions in the UniformMu population, request seeds, generate segregating families, PCR-genotype seedlings for Mu insertions of interest, and associate genotypes with phenotypes. Resulting analyses provide definitive, in planta evidence for genotype-phenotype relationships that either support or refute hypotheses regarding gene function.

Geneticists frequently use loss-of-function (knockout) mutations to reveal the effects of a gene's dysfunction at the organismal level, observed as the mutant phenotype. This strategy is facilitated by creation of large, searchable collections of knockout mutants in an organism of interest. Paramount among such resources in maize is the UniformMu National Resource, a large collection of genetic stocks carrying mutations generated by insertions of Robertson's Mutator (Mu) transposons. The name UniformMu refers to the phenotypic uniformity of the W22 inbred genetic background in which Mu insertion mutants were created. This community resource continues its pivotal role in providing seeds containing beneficial knockout and knockdown mutations in targeted genes, which can be used to elucidate gene function. The resource offers an invaluable complement to other functional genomics approaches aimed at bridging the gap between genome sequences and plant performance in the field. Several key features are central to the success of the UniformMu National Public Resource. First, mapped insertions are linked to seed stocks that are readily available through the Maize Genetics and Genomics Database (MaizeGDB) and the Maize Genetics Cooperation Stock Center. Second, a uniform inbred background facilitates analysis of mutant phenotypes, by providing uniform wild-type controls. Third, mutant alleles are reliably heritable and consistently recovered in stated lines. Finally, lines are stable, with no continuing transposition of Mu insertions. The collective effort of the maize community allows UniformMu to provide readily accessible knockout and knockdown mutant seeds, as well as, ultimately, highly sought evidence for gene function in planta.

To understand what drives an immune response, it is important to characterize, at a molecular level, the site(s) on an immunogenic antigen that is directly contacted by a soluble antibody or B-cell antigen receptor (BCR) on the surface of a B lymphocyte. Moreover, antibody binding interactions with a microbial protein can interfere with the functional activity of a toxin (i.e., neutralization) and/or can aid in the clearance of the microbial protein from the body, further underscoring the importance of such characterization. Phage display technology is a potent tool that can be used to study any type of protein-protein interaction. In recent years, we have refined methods for the identification of the minimal binding contact sites of an antibody with an antigen. Here, we describe a workflow for optimizing antibody-mediated selection and for the identification and characterization of antigen-specific epitopes. This workflow includes (1) the generation of large libraries of random fragments of a gene of interest cloned into the validated pComb-Opti8 phagemid expression cloning vector system; (2) electroporation of these libraries into electrocompetent bacterial cells and subsequent recovery of viral particles, each of which displays the cloned gene fragment product as a fusion protein with the filamentous phage major coat protein VIII (pVIII); (3) recovery of individual phagemid clones that express the smallest functional epitopes recognized by an experimental antibody; (4) an efficient means of using high-throughput DNA sequencing to interrogate sequentially selected libraries to rapidly identify the gene subregions encoding epitopes of interest; and (5) means for the further characterization of potential antibody-epitope binding interactions.