Bio-protocol最新文献

Labeling cells with reporter genes allows researchers to visually identify specific cells and observe how they interact with each other in dynamic biological systems. Even though various labeling methods are now available, a specific description of gene knock-in labeling methods for human trophoblast stem cells (hTSCs) has not been reported. Here, we present a streamlined protocol for labeling hTSCs with the green fluorescent protein (GFP) reporter gene via CRISPR/Cas9-mediated knock-in of the gene into the adeno-associated virus site 1 (AAVS1) safe harbor locus. A commonly used hTSC cell line, CT29, was transfected with a dual plasmid system encoding the Cas9 endonuclease and an AAVS1-targeted guide RNA in one plasmid and a donor plasmid encoding a puromycin resistance gene and GFP reporter gene flanked by AAVS1 homology arms. Puromycin-resistant clonal cells were isolated, and AAVS1 integration was confirmed via PCR and sequencing of the PCR products. The labeled cells are proliferative and can give rise to extravillous cytotrophoblast cells (EVT) and the syncytiotrophoblast (ST). To our knowledge, this is the first report using the CRISPR/Cas9 system for AAVS1 integration of a reporter gene in human trophoblast stem cells. It provides an efficient tool to facilitate the study of human trophoblast development and function in co-culture systems and will be highly useful in developing clinical gene therapy-related plasmid constructs. Key features • First report to constitutively express a fluorescent label in hTSCs by applying a CRISPR/Cas9 knock-in approach and an AAVS1 safe harbor locus. • Provides an efficient tool to facilitate the study of human trophoblast development and function, particularly in heterologous co-culture systems. • Offers an approach for developing clinical gene therapy-related plasmid constructs that allow insertion of therapeutic genes without associated disruption of essential genes. • Widely applicable approach to label other human cell lines.

Traditional methods for studying protein-protein interactions often lack the resolution to quantitatively distinguish distinct oligomeric states, particularly for membrane proteins within their native lipid environments. To address this limitation, we developed SiMPull-POP (single-molecule pull-down polymeric nanodisc photobleaching), a single-molecule technique designed to quantify membrane protein oligomerization with high sensitivity and in a near-native context. The goal of SiMPull-POP is to enable precise, quantitative analysis of membrane protein assembly by preserving native lipid interactions using diisobutylene maleic acid (DIBMA) to form nanodiscs. Unlike ensemble methods such as co-immunoprecipitation or FRET, which average out heterogeneous populations, SiMPull-POP uses photobleaching to resolve monomeric, dimeric, and higher-order oligomeric states at the single-molecule level. We validated SiMPull-POP using several model systems. A truncated, single-pass transmembrane protein (Omp25) appeared primarily monomeric, while a membrane-tethered FKBP protein exhibited ligand-dependent dimerization upon addition of the AP ligand. Applying SiMPull-POP to EphA2, a receptor tyrosine kinase, we found it to be mostly monomeric in the absence of its ligand, Ephrin-A1, and shifting toward higher-order oligomers upon ligand binding. To explore factors influencing ligand-independent assembly, we modulated membrane cholesterol content. Reducing cholesterol induced spontaneous EphA2 oligomerization, indicating that cholesterol suppresses receptor self-association. Overall, SiMPull-POP offers significant advantages over conventional techniques by enabling quantitative, single-molecule resolution of membrane protein complexes in a native-like environment. This approach provides critical insights into how membrane properties and external stimuli regulate protein assembly, supporting broader efforts to understand membrane protein function in both normal and disease states. Key features • Precise determination of membrane protein stoichiometry (e.g., monomer, dimer, oligomer) by directly counting photobleaching steps, overcoming the averaging limitations of bulk assays. • By incorporating membrane proteins into DIBMA lipid particles (DIBMALPs), this preserves native lipid interactions, offering a more physiologically relevant context for studying protein assembly. • Sensitively detects ligand-induced or membrane property-driven changes in oligomerization, making it a powerful tool for investigating both constitutive and regulated protein interactions.

In recent years, the calcifying properties of some cyanobacteria have been used in the production of living building materials (LBMs), such as bio-concrete, as a CO₂-friendly alternative for cement. This microbially induced calcium carbonate precipitation (MICP) technique can act as a novel platform technology for carbon capture strategies. Consequently, various research articles have been conducted based on a diverse set of workflows, including several modifications, to manufacture LBMs. However, such articles contain only fragmentary descriptions of the materials and methods used. This protocol is meant to act as a detailed, step-by-step operational manual for the production of LBMs using the cyanobacterial model strain Picosynechococcus sp. PCC 7002. The process is divided into several steps, such as the activation of the cyanobacterial-gel solution with CaCl₂ × 2H₂O and NaHCO₃, casting standardized prisms (160 × 40 × 40 mm), and demolding LBMs. Subsequently, bending tensile and compressive strength tests are performed according to the procedures commonly used in concrete and material testing as proof of concept. Key features • A comprehensive workflow for the manufacturing of cement-free living building materials with cyanobacteria. • A cyanobacteria-gelatin-containing solution is activated, mixed with sand, casted, curated, and strength tested. • Adaptable for other cyanobacterial strains and substitute materials.

Understanding how lipids interact with lipid transfer proteins (LTPs) is essential for uncovering their molecular mechanisms. Yet, many available LTP structures, particularly those thought to function as membrane bridges, lack detailed information on where their native lipid ligands are located. Computational strategies, such as docking or AI-methods, offer a valuable alternative to overcome this gap, but their effectiveness is often restricted by the inherent flexibility of lipid molecules and the lack of large training sets with structures of proteins bound to lipids. To tackle this issue, we introduce a reproducible computational pipeline that uses unbiased coarse-grained molecular dynamics (CG-MD) simulations on a free and open-source software (GROMACS) with the Martini 3 force-field. Starting from a configuration of a lipid in bulk solvent, we run CG-MD simulations and observe spontaneous binding of the lipid to the protein. We show that this protocol reliably identifies lipid-binding pockets in LTPs and, unlike docking methods, suggests potential entry routes for lipid molecules with no a priori knowledge other than the protein's structure. We demonstrate the utility of this approach in investigating bridge LTPs whose internal lipid-binding positions remain unresolved. Altogether, our study provides a cost-effective, efficient, and accurate framework for mapping binding sites and entry pathways in diverse LTPs. Key features • Demonstrates the reliability of unbiased coarse-grain molecular dynamics (CG-MD) simulations with the Martini 3 force-field in identifying lipid-binding sites in lipid transfer proteins (LTPs). • The protocol is straightforward to replicate, relying solely on freely available open-source software. • Furthermore, it is computationally efficient, with most simulations completing within a few hours on a standalone GPU-accelerated workstation. • As input, the user only needs to include the structure of the protein and select the lipid type to test.

This protocol presents a modified version of the Filterprep method originally reported in New Biotechnology, adding an optional step to reduce endotoxin levels. Filterprep is a simple, rapid, and cost-effective approach to plasmid DNA purification that couples ethanol precipitation with a single spin-column filtration step, eliminating chaotropic salts and silica binding. The formulations and parameters are fully transparent and do not rely on proprietary buffers, using only standard laboratory reagents and widely available miniprep columns. Under matched conditions, the method recovers high-purity plasmid DNA with yields up to fivefold higher than those obtained with representative commercial midiprep kits. The workflow is readily adoptable in most molecular biology laboratories and, under routine conditions, can be completed in approximately 40 min. The resulting DNA is suitable for molecular cloning, PCR, sequencing, and other downstream biochemical applications. Endotoxin is a lipopolysaccharide (LPS) found in the outer membrane of Gram-negative bacteria and may carry over during plasmid preparation. For experiments requiring lower endotoxin input, an optional modification resuspends the DNA pellet in a Triton X-114 wash buffer before column loading to decrease lipopolysaccharide carryover. The method is modular and extensible, allowing adjustment of precipitation and wash conditions, variation in the number of washes, selection of alternative column formats, and integration of endotoxin-reduction modules without altering the core principle. These features facilitate troubleshooting and quality control, enable scaling from routine batches to larger culture volumes and higher throughput, and allow seamless integration with existing workflows. Key features • Modular, chaotrope-free workflow [1] combining ethanol precipitation and single-column cleanup; transparent chemistry allows RNase, endotoxin reduction, or extra washes without changing the core principle. • Uses only standard reagents, a microcentrifuge, and common miniprep columns, with no proprietary kits, vacuum manifolds, or specialized equipment, enabling broad adoption across laboratories. • Extensible across scales from small miniprep volumes to larger cultures while remaining compatible with cloning, PCR, sequencing, and transfection-grade applications. • Optional low-endotoxin modification resuspends the DNA pellet in Triton X-114 wash buffer before column filtration to reduce lipopolysaccharide carryover.

Hair cells are the sensory receptors of the auditory and vestibular systems in the inner ears of all vertebrates. Hair cells also serve to detect water flow in the lateral line system in amphibians and fish. The zebrafish lateral line serves as a well-established model for investigating hair cell development and function, including research on genetic mutations associated with deafness and environmental factors that cause hair cell damage. Rheotaxis, the ability to orient and swim in response to water flow, is a behavior mediated by multiple sensory modalities, including the lateral line organ. In this protocol, we describe a rheotaxis assay in which station holding behavior, which employs positive rheotaxis to maintain position in oncoming water flow, serves as a sensitive measure of lateral line function in larval zebrafish. This assay provides a valuable tool for researchers assessing the functional consequences of genetic or environmental disruptions of the lateral line system. Key features • Describes the method developed by Newton et al. [1] to assess lateral line function in larval zebrafish. • Provides instructions on building the micro flume apparatus with updated information from the WashU Neurotech Hub. • Uses DeepLabCut to track fish and SimBA to classify rheotaxis to compare lateral line-mediated behaviors in large cohorts of larval zebrafish.

Plants move chloroplasts in response to light, changing the optical properties of leaves. Low irradiance induces chloroplast accumulation, while high irradiance triggers chloroplast avoidance. Chloroplast movements may be monitored through changes in leaf transmittance and reflectance, typically in red light. We present a step-by-step procedure for the detection of chloroplast positioning using reflectance hyperspectral imaging in white light. We show how to employ machine learning methods to classify leaves according to the chloroplast positioning. The convolutional network is a method of choice for the analysis of the reflectance spectra, as it allows low levels of misclassification. As a complementary approach, we propose a vegetation index, called the Chloroplast Movement Index (CMI), which is sensitive to chloroplast positioning. Our method offers a high-throughput, contactless way of chloroplast movement detection. Key features • Protocol for detached leaves handled in laboratory conditions. • Based on differential (dark-adapted versus irradiated) hyperspectral images of plant leaves. • Data analysis includes machine learning methods and the calculation of a vegetation index. • Requires irradiation equipment apart from the hyperspectral camera set.

Optogenetic stimulation of peripheral motor nerves is a promising technique for modulating neural activity via illumination of light-sensitive ion channels known as opsins. Stimulating muscle activity through this method offers many advantages, such as a physiological recruitment order of motor units, reduced fatigue, and target-specific stimulation, which make it a favorable option for use in many neuroscience and motor rehabilitation applications. To enable such optical stimulation, opsin expression in peripheral nerves can be achieved either with transgenic animal models or through injection of viral vectors. In this protocol, we describe a method for driving peripheral nerve opsin expression via intramuscular adeno-associated virus (AAV) injection with the goal of enhancing virus uptake by targeting injections to neuromuscular junctions with electrical stimulation. We also describe procedures for non-invasively assessing functional opsin expression over time with transdermal optical stimulation of opsin-labeled nerves and electromyography (EMG) recordings. The presence of time-locked EMG spikes 4-8 ms after each stimulation pulse demonstrates that functional opsin expression is present at a given assessment time point. Onset of functional optical sensitivity generally occurs 2-4 weeks following virus injection, and sensitivity generally peaks or plateaus between 6-10 weeks. Stimulation sequences such as light intensity, stimulation pulse width, and frequency sweeps provide further information on functional opsin expression at the testing timepoint. The methods presented here can be used for driving functional opsin expression with a standard AAV6 vector commonly used in similar experiments or as a protocol for assessing peripheral nerve opsin expression with novel viral vectors. Key features • Uses electrical stimulation to guide needle placement during intramuscular viral injection. • Drives robust and muscle-specific opsin expression in peripheral motor neurons. • Describes transdermal optical stimulation sequences with varying stimulation light intensity, pulse width, and frequency for longitudinal assessment of opsin expression. • Adaptable for use with multiple viral vectors and target muscles.

A simple and effective method to identify genetic markers of yield response to nitrogen (N) fertilizer among maize hybrids is urgently needed. In this article, we describe a detailed methodology to identify genetic markers and develop associated assays for the prediction of yield N-response in maize. We first outline an in silico workflow to identify high-priority single-nucleotide polymorphism (SNP) markers from genome-wide association studies (GWAS). We then describe a detailed methodology to develop cleaved amplified polymorphic sequences (CAPS) and derived CAPS (dCAPS)-based assays to quickly and effectively test genetic marker subsets. This protocol is expected to provide a robust approach to determine N-response type among maize germplasm, including elite commercial varieties, allowing more appropriate on-farm N application rates, minimizing N fertilizer waste. Key features • Leverages GWAS datasets to efficiently identify genetic markers. • Employs basic molecular biology techniques. • Can be adapted to any maize germplasm.

In mammals, the semen is ejaculated into the female reproductive tract, and the sperm travel to the oviduct to fertilize the egg. A comprehensive understanding of the pre- and post-ejaculatory intrauterine environment is one of the key points for overcoming infertility; however, the dynamics of the intrauterine environment and its physiological role in the uterus, namely in the internal fertilization process, remain unclear. Conventional methods for collecting uterine fluids from the uterus post-ejaculation of mice show challenges regarding the ambiguous ejaculation timing. Here, we established a method for a mating environment with exact ejaculation timing. We also created a simple method for collecting pre- and post-ejaculatory uterine fluid without using forceps. Our methods achieved time-dependent biochemical and histological analyses of uterine fluids to provide fundamental information regarding protein composition and uterine structure changes during pre- and post-ejaculation. This protocol is suitable for analyzing temporal changes in reproductive phenomena, thereby contributing to elucidating the physiological role of the uterus in the process of intrauterine fertilization. Key features • This protocol is used for the simple collection of pre- and post-ejaculatory uterine fluid. • Changes in the pre- and post-ejaculatory intrauterine environment can be examined by controlling the dissection time of females after ejaculation. • An estrous female can be determined without a vaginal smear test in this protocol. • This protocol can be used to analyze the protein composition of post-ejaculatory uterine fluid and is applicable to analyze sperm within the uterus post-ejaculation.