Bio-protocol最新文献

Genetic modification is essential for understanding parasite biology, yet it remains challenging in Plasmodium. This is partially due to the parasite's low genetic tractability and reliance on homologous recombination, since the parasites lack the canonical non-homologous end-joining pathway. Existing approaches, such as the PlasmoGEM project, enable genome-wide knockouts but remain limited in coverage and flexibility. Here, we present the Plasmodium berghei high-throughput (PbHiT) system, a scalable CRISPR-Cas9 protocol for efficient genome editing in rodent malaria parasites. The PbHiT method uses a single cloning step to generate vectors in which a guide RNA (gRNA) is physically linked to short (100 bp) homology arms, enabling precise integration at the target locus upon transfection. The gRNA also serves as a unique barcode, allowing pooled vector transfections and identification of mutants by downstream gRNA sequencing. The PbHiT system reliably recapitulates known mutant growth phenotypes and supports both knockout and tagging strategies. This protocol provides a reproducible and scalable tool for genome editing in P. berghei, enabling both targeted functional studies and high-throughput genetic screens. Additionally, we provide an online resource covering the entire P. berghei protein-coding genome and describe a step-by-step pooled ligation approach for large-scale vector production. Key features • PbHiT provides a high-throughput CRISPR-Cas9 genome editing platform optimised for Plasmodium berghei experimental infections in rodents. • This protocol enables efficient and reproducible generation of knockout and tagged parasite lines using short homology arms. • This protocol is supported by a free online resource for P. berghei gene construct design and requires basic knowledge of cloning. • Transfection of Plasmodium berghei requires experience in handling mice/rats, an ethical permit, and an animal facility.

Underwater noise is a growing source of anthropogenic pollution in aquatic environments. However, few studies have evaluated the impact of underwater noise on aquatic invertebrates. More importantly, studies involving early developmental stages have been poorly addressed. Significant limitations are due to the lack of standardized protocols for working in the laboratory. Particularly, the design of uniform procedures in the laboratory is important when working with species that inhabit short-term changing habitats, such as estuaries, which makes it difficult to carry out repeated experiments in the natural habitat. Besides, controlling for environmental variables is also important when assessing the effect of a stressor on the physiological parameters of individuals. This experimental protocol addresses that gap by offering an adaptable laboratory-based method to evaluate sublethal physiological responses to sound exposure under highly controlled conditions. Here, we present a reproducible and accessible laboratory protocol to expose crabs to recorded boat noise and evaluate physiological responses using oxidative stress biomarkers. The method is designed for ovigerous females, as we evaluated the effects on embryos and early life stages (i.e., larvae), but it can be readily adapted to different life stages of aquatic invertebrates. A key strength of this protocol is its simplicity and flexibility: animals are exposed to noise using submerged transducers under well-controlled laboratory conditions, ensuring consistency and repeatability. Following exposure, tissues or whole-body samples can be processed for a suite of oxidative stress biomarkers-glutathione-S-transferase (GST), catalase (CAT), lipid peroxidation (LPO), and protein oxidation. These biomarkers are highly responsive, cost-effective indicators that provide a sensitive and early readout of sublethal stress. Together, the exposure and analysis steps described in this protocol offer a powerful and scalable approach for investigating the physiological impacts of underwater noise in crustaceans and other aquatic invertebrates. Key features • Enables measurement of oxidative stress markers across different life stages-from embryos to larvae and adult tissues-offering a complete view of physiological impact. • Ensures consistent, reproducible conditions through standardized exposure and sampling, supporting reliable comparisons across experiments. • Flexible protocol adaptable to Neohelice granulata and other estuarine decapods or marine benthic invertebrates, broadening its applicability.

To study gene function in regulating rodent retinal waves during development, an efficient method for gene delivery into whole-mount retinas is required while preserving circuit functionality for physiological studies. We present an optimized electroporation protocol for developing rodent retinal explants. The procedure includes the fabrication of horizontally aligned platinum electrodes and the placement of retinal explants between them to generate a uniform electric field for high transfection efficiency. The entire process-dissection and electroporation-can be completed within 1-2 h. Successful transfection is verified by fluorescence microscopy, and physiological assays such as patch-clamp recordings and live imaging can be performed within 1-4 days following electroporation. This rapid and reliable protocol enables functional analysis for a specific gene in regulating retinal waves and can be adapted to other organotypic slice cultures. Key features • Incorporates horizontally aligned platinum electrodes and enables cell type-specific promoters to drive gene expression for physiological studies. • Preserves retinal wave activity while markedly improving transfection efficiency in whole-mount postnatal rodent retinas. • Requires only 1-2 h from retinal dissection to electroporation. • Allows completion of functional experiments within four days after electroporation.

Expansion microscopy (ExM) is an innovative and cost-effective super-resolution imaging technique that enables nanoscale visualization of biological structures using conventional fluorescence microscopes. By physically enlarging biological specimens, ExM circumvents the diffraction limit and has become an indispensable tool in cell biology. Ongoing methodological advances have further enhanced its spatial resolution, labeling versatility, and compatibility with diverse sample types. However, ExM imaging is often hindered by sample drift during image acquisition, caused by subtle movements of the expanded hydrogel. This drift can distort three-dimensional reconstruction, compromising both visualization accuracy and quantitative analysis. To overcome this limitation, we developed 3D-Aligner, an advanced and user-friendly image analysis software that computationally corrects sample drift in fluorescence microscopy datasets, including but not limited to those acquired using ExM. The algorithm accurately determines drift trajectories across image stacks by detecting and matching stable background features, enabling nanometer-scale alignment to restore structural fidelity. We demonstrate that 3D-Aligner robustly corrects drift across ExM datasets with varying expansion factors and fluorescent labels. This protocol provides a comprehensive, step-by-step workflow for implementing drift correction in ExM datasets, ensuring reliable three-dimensional imaging and quantitative assessment. Key features • 3D-Aligner precisely corrects sample drift in expansion microscopy (ExM) datasets, enabling reliable 3D reconstruction and robust quantitative analysis. • Utilizes background feature detection and feature matching across z-planes to achieve nanoscale-precision drift correction. • 3D-Speckler, which is a MATLAB-based software platform, offers a customizable and user-friendly interface. • Outperforms conventional registration tools across varying expansion factors and labeling conditions and is equally applicable to non-ExM datasets.

Accurate profiling of soil and root-associated bacterial communities is essential for understanding ecosystem functions and improving sustainable agricultural practices. Here, a comprehensive, modular workflow is presented for the analysis of full-length 16S rRNA gene amplicons generated with Oxford Nanopore long-read sequencing. The protocol integrates four standardized steps: (i) quality assessment and filtering of raw reads with NanoPlot and NanoFilt, (ii) removal of plant organelle contamination using a curated Viridiplantae Kraken2 database, (iii) species-level taxonomic assignment with Emu, and (iv) downstream ecological analyses, including rarefaction, diversity metrics, and functional inference. Leveraging high-performance computing resources, the workflow enables parallel processing of large datasets, rigorous contamination control, and reproducible execution across environments. The pipeline's efficiency is demonstrated on full-length 16S rRNA gene datasets from yellow pea rhizosphere and root samples, with high post-filter read retention and high-resolution community profiles. Automated SLURM scripts and detailed documentation are provided in a public GitHub repository (https://github.com/henrimdias/emu-microbiome-HPC; release v1.0.2, emu-pipeline-revised) and archived on Zenodo (DOI: 10.5281/zenodo.17764933). Key features • Implement rigorous quality control (QC) of raw 16S rRNA Nanopore reads and sequencing controls. • Remove plant organelle contamination with a curated Kraken2 database. • Perform high-resolution taxonomic assignment of full-length 16S rRNA reads using Emu. • Integrate downstream statistical analyses, including rarefaction, PERMANOVA, and DESeq2 differential abundance. • Conduct scalable microbiome diversity and functional analyses with FAPROTAX.

Adipogenic differentiation efficiency remains highly variable across laboratories and cellular models, underscoring a critical need for a robust and standardized protocol. Here, we describe an optimized and highly effective protocol for inducing adipogenesis in multiple models, including murine 3T3-L1 preadipocytes, stromal vascular fraction (SVF) from neonatal and adult mice, and human adipose-derived stem cells (hADSCs). Systematic optimization was performed on key parameters such as initial cell confluence, induction timing, inducer composition, and culture surface coating. We show that high cell density, rosiglitazone supplementation, and an extended primary induction phase combine to promote lipid accumulation. Notably, we introduce a crucial modification-prolonged low-dose insulin stimulation during the maintenance phase-that is essential for the efficient differentiation of adult SVF. Furthermore, when applied to hADSCs, the protocol consistently induced robust adipogenesis, confirming its cross-species applicability. Taken together, this comprehensive and reproducible protocol serves as a valuable tool for advancing in vitro adipogenesis research. Key features • Extend a robust, standardized adipogenic differentiation protocol from 3T3-L1 preadipocytes to clinically relevant models, including hADSCs and the heterogeneous SVF. • Identify key optimized parameters-cell density, induction timing, and inducer composition-enabling highly reproducible differentiation across species.

Congenital renal disorders, such as the Potter sequence, result from renal dysgenesis. To explore a prenatal therapeutic approach for fetuses with kidney insufficiency, we established an in utero transplantation protocol using donor fetal kidneys. Although numerous rodent studies have reported cellular injections into fetal recipients, no protocol to date has described whole-organ transplantation during gestation. Here, we present a step-by-step method for grafting donor fetal kidneys (embryonic day 14.0-16.5) into allogeneic rat fetuses at embryonic day 18.0-18.5, resulting in term neonates that retain the grafts postnatally. A 15-16 G needle preloaded with the donor kidney is inserted transuterinely, depositing the organ into the subcutaneous space of the fetus. Four days later, the term pups are delivered naturally and evaluated for graft development. This protocol enables organ-level transplantation and longitudinal assessment of graft maturation within the unique fetal environment, which differs markedly from adult settings in terms of growth factor availability and immune reactivity. To our knowledge, this is the first protocol to successfully achieve whole-organ transplantation directly into fetuses in utero. Therefore, the model provides a valuable platform for studying developmental organogenesis, fetal immunology, and regenerative strategies that leverage embryonic cues. Key features • Subcutaneous transplantation of fetal kidneys into recipient fetuses minimizes surgical invasiveness and significantly improves fetal survival. • Natural delivery enables pups to nurse from the dam, allowing extended postnatal observation. • Use of green fluorescent protein (GFP)-expressing donor tissue permits real-time visualization of graft location and growth. • The protocol is readily adaptable for xenotransplantation and studies of immunological tolerance during fetal development.

Although protein-protein interactions (PPIs) are central to nearly all biological processes, identifying and engineering high-affinity intracellular binders remains a significant challenge due to the complexity of the cellular environment and the folding constraints of proteins. Here, we present a two-stage complementary platform that combines magnetic-activated cell sorting (MACS)-based yeast surface display with functional ligand-binding identification by twin-arginine translocation (Tat)-based recognition of associating proteins (FLI-TRAP), a bacterial genetic selection system for efficient screening, validation, and optimization of PPIs. In the first stage, MACS-based yeast display enables the rapid high-throughput identification of candidate binders for a target antigen from a large synthetic-yeast display library through extracellular interaction screening. In the second stage, an antigen-focused library is subcloned into the FLI-TRAP system, which exploits the hitchhiker export process of the Escherichia coli Tat pathway to evaluate binder-antigen binding in the cytoplasm. This stage is achieved by co-expressing a Tat signal peptide-tagged protein of interest with a β-lactamase-tagged antigen target, such that only binder-antigen pairs with sufficient affinity are co-translocated into the periplasm, thus rendering the bacterium β-lactam antibiotic resistant. Because Tat-dependent export requires fully folded and soluble proteins, FLI-TRAP further serves as a stringent in vivo filter for intracellular compatibility, folding, and stability. Therefore, this approach provides a powerful and cost-effective pipeline for discovering and engineering intracellular protein binders with high affinity, specificity, and functional expression in bacterial systems. This workflow holds promise for several applications, including synthetic biology and screening of theragnostic proteins and PPI inhibitors. Key features • Combines a single round of MACS enrichment with FLI-TRAP for high-throughput Nb discovery. • Reduces time and resource demands compared to traditional workflows involving multiple rounds of MACS/FACS. • Enables in vivo selection of high-affinity, functional binders via Tat-dependent export linked to β-lactam resistance, correlating binding affinity and solubility with antibiotic resistance.

Toxoplasma gondii is an apicomplexan parasite that infects a wide variety of eukaryotic hosts and causes toxoplasmosis. The cell cycle of T. gondii exhibits a distinct architecture and regulation that differ significantly from those observed in well-studied eukaryotic models. To better understand the tachyzoite cell cycle, we developed a fluorescent ubiquitination-based cell cycle indicator (FUCCI) system that enables real-time visualization and quantitative assessment of the different cell cycle phases via immunofluorescence microscopy. Quantitative immunofluorescence and live-cell imaging of the ToxoFUCCI^S probe with specific cell cycle markers revealed substantial overlap between cell cycle phases S, G₂, mitosis, and cytokinesis, further confirming the intricacy of the apicomplexan cell cycle. Key features • This protocol describes the development of the transgenic lines capable of detecting individual cell cycle phases and processes of the Toxoplasma tachyzoite cell cycle. • Quantitative immunofluorescence analysis and real-time microscopy enable the measurement of each cell cycle phase. • The ToxoFUCCI^S probe helps to gain new insights into the highly flexible, overlapping nature of cell cycle organization in apicomplexan parasites.

It is common practice for laboratories to discard clotted blood or freeze it for future DNA extraction after extracting serum from a serum-separating tube. If freezing for DNA extraction, the blood clot is not usually cryopreserved, which leads to cell membrane fragility. In this protocol, we describe steps to isolate high-quality nuclei from leukocytes derived from whole blood samples frozen without a cryoprotective medium. Nuclei isolated from this protocol were able to undergo ATAC (assay for transposase-accessible chromatin) sequencing to obtain chromatin accessibility data. We successfully characterized and isolated B cells and T cells from leukocytes isolated from previously frozen blood clot using Miltenyi's gentleMACS Octo Dissociator coupled with flow sorting. Nuclei showed round, intact nuclear envelopes suitable for downstream applications, including bulk sequencing of nuclei or single-cell nuclei sequencing. We validated this protocol by performing bulk ATAC-seq. Key features • This protocol is compatible with previously collected blood that has been frozen. • Previous cryopreservation of the samples is not required for this protocol. • This protocol enables flow sorting of non-viable leukocytes for a more precise cell population for bulk sequencing experiments.