Methods in molecular biology最新文献

The zebrafish is a valuable animal model for investigating the genetic basis of vertebrate evolution, development, behavior, and regeneration. However, the existence of numerous gene paralogs in the zebrafish genome represents a major challenge, complicating functional genomic research using reverse-genetics approaches. To facilitate reverse genetics-based phenotypic screens, we recently presented simple methods that enable efficient induction of biallelic gene disruptions in F0 zebrafish, providing a rapid avenue for screening potential gene functions through consistent phenotypic detection. Here, we describe detailed protocols for these CRISPR/Cas9-based mutagenesis strategies to achieve highly effective biallelic gene inactivation in F0 zebrafish. The high consistency of these strategies, combined with a streamlined workflow, offers a robust phenotypic screening platform for a quick and reliable functional assessment of genes of interest, both individually and in a scalable manner. These strategies enhance the efficacy of successful F0 zebrafish phenotypic screening, thereby accelerating functional genetic studies using this powerful model organism.

The nematode Caenorhabditis elegans is a eukaryotic genetic model organism introduced for studies of animal development and behavior (Brenner S, Genetics 77:71-94, 1974). It is also proving useful to expedite our understanding of human diseases and to explore potential therapies (Ahringer J, Curr Opin Genet Dev 7:410-415, 1997; Culetto E, Sattelle DB, Hum Mol Genet 9:869-877, 2000). Monitoring phenotypic changes and the impact of drug candidates is particularly convenient in the case of C. elegans models of neuromuscular or neurological disorders, where changes in motility and growth are often easily observed and can be conveniently assayed. We therefore developed an Invertebrate Automated Phenotyping Platform (INVAPP) together with an algorithm (Paragon) to facilitate such work (Buckingham SD, Partridge FA, Sattelle DB, Int J Parasitol Drugs Drug Resist Int J Parasitol Drugs Drug Resist 4:226-232, 2014; Partridge FA, Brown AE, Buckingham SD, Willis NJ, Wynne GM, Forman R et al., Int J Parasitol Drugs Drug Resist 8:8-21, 2018). Similarly, in the search for novel chemicals to combat invertebrate pathogens, such as parasitic worms, and disease vectors, such as the mosquito that serves as the malaria parasite vector, the phenotyping of worms and insects in the presence of new candidate drugs and control chemicals (anthelmintics and insecticides) can be extremely useful. This is especially important in view of the current challenges in controlling the malaria vector Anopheles gambiae and the soil-transmitted helminth, the whipworm Trichuris trichiura. For example, the development of resistance to the hitherto highly successful pyrethroid insecticides threatens the impressive gains made by the deployment of insecticide-treated nets (ITNs) and indoor residual sprays (IRS) in reducing malaria cases in the period 2000-2015. Also, there is a need for new anthelmintic drugs to combat soil-transmitted helminths such as whipworm, now that the widely used benzimidazoles are becoming much less effective. In both cases, automated phenotyping assays have a role to play. Here, we describe the use of a simple invertebrate automated phenotyping system and provide some examples that illustrate its utility.

The kinetic stability of peptides bound onto MHC class I molecules is a critical parameter that helps shape their interaction with immune receptors and consequently their antigenic potential. We present here a method for measuring the kinetic stability and sensitivity to proteolytic degradation of peptides bound onto MHC class I molecules after in vitro refolding, by analyzing the time-resolved MALDI-TOF Mass Spec signal from the peptide, using the whole MHC-I/peptide complex in situ. This approach can provide important information on the dynamic nature of the MHC-peptide interaction, the kinetic half-life of binding and the sensitivity of the peptide to external proteolytic digestion or other modifications.

There are various ways to activate human T cells through their T Cell Receptor (TCR) or through overexpressed Chimeric Antigen Receptors (CARs), which are one of the most promising treatments in cancer immunotherapy. Here, we describe some basic methods to activate the human TCR or CAR using Antigen Presenting Cells presenting their natural antigen or superantigen, or stimulation with soluble antibodies or antibody-covered beads. There are several methods to analyze this activation and we describe here cytokine detection by ELISA, phosphoprotein detection by Western blot and expression of activation molecules at the cell surface by flow cytometry.

T cells are key players of the cellular immune system, able to detect and fight pathogens' invasion. T cells recognize pathogen-derived peptides presented by molecules called Human Leukocyte Antigens (HLA) in humans. The interaction between the T cells and peptide-HLA (pHLA) complexes is driven by the surface T cell receptors (TCRs). This interaction is critical and the first step of T cell activation. The affinity of the TCR for the pHLA is one of the key drivers of T cell activation. In this chapter, we describe the method to determine the affinity of the TCR for the pHLA via surface plasmon resonance.

Endoplasmic reticulum aminopeptidases 1 and 2 (ERAP1 and ERAP2) play a critical role in antigen processing by trimming N-terminally extended peptides within the ER, thereby shaping the repertoire of peptides presented by Major Histocompatibility Complex Class I (MHC I) molecules. Polymorphic variants in these enzymes give rise to functionally distinct allotypes, influencing peptide trimming efficiency and specificity, modulating CD8+ T cell responses in both health and disease. This chapter outlines a cellular model system for assessing ERAP1 and ERAP2 peptide trimming activity, using peptide-specific T cell activation as a surrogate readout. By employing transient transfection and co-culture with either T cell hybridoma (B3Z) or cytotoxic T lymphocytes (CTL), this approach enables the evaluation of peptide processing efficiency of ERAP1/2 based on the presentation and recognition of optimally generated MHC I-restricted epitopes.

Spiroplasma melliferum is a cultivable mollicute that can be used as a proxy model organism to test the inhibitory effects of filtrates, compounds, or substances against phytoplasma. In this protocol, we describe the use of the resazurin-based alamarBlue™ dye for the monitoring of Spiroplasma survival and growth in the presence of presumably inhibitory agents.

In this introductory chapter, we provide an overview of phytoplasma biology and outline the historical milestones that shaped the field, from their first discovery to their current taxonomic status. We also highlight how this third edition of "Phytoplasmas: Methods and Protocols" differs from previous volumes, reflecting both the adoption of new molecular and sequencing technologies and the revival of classical approaches. Taken together, these developments illustrate the evolving trajectory of phytoplasma research and set the stage for the detailed protocols presented in the following chapters.

Grapevine is among the most important economic plants that are susceptible to phytoplasma disease. Tools and procedures for genetic manipulation of grapevine are cumbersome and sometimes difficult to replicate; therefore, most of the research on plant-phytoplasma interactions focuses on species with more established transformation procedures instead, even though they might not be hosts of the studied phytoplasma. Studying the mechanisms of plant-phytoplasma interactions directly in the host plants would bring new and more accurate insights into the disease mechanisms. In this chapter, we present the protocols to obtain transformation-competent grapevine material from different tissues and the procedures for protoplast isolation and cultivation.

Plant adaptive responses to their environment are orchestrated by transcriptional reprogramming, regulated by sequence-specific transcription factors (TFs) recognizing specific TF-binding sites (TFBSs), and understanding their regulatory grammar is crucial for unravelling plant adaptation mechanisms. Empirical data have contributed to the elucidation of TFBS sequence motifs involved in biological processes, but comprehensive experimental approaches may not be feasible, especially in non-model species. Different computational algorithms have facilitated the elucidation of TFBS sequence motifs and the construction of predictive models, shedding light on plant gene regulatory networks. In this context, the development of straightforward computational pipelines and easy-to-use bioinformatics tools is of particular relevance to make gene expression analysis accessible to the research community. This chapter presents a methodology to infer TF regulators applicable to 60 plant species from RNA sequencing (RNA-seq) data as the starting point. It includes RNA-seq analysis and quantification, gene clustering using WGCNA to identify co-regulated gene modules, and searching for enriched TFBSs associated with these modules. The methodology is illustrated using Arabidopsis RNA-seq data related to abiotic stress. By providing a user-friendly pipeline, researchers are empowered to unravel the molecular basis of gene expression dynamics in plants.