Bio-protocol最新文献

{"title":"High-Throughput Screening Identification of Chemical Compounds That Affect Cold-Regulated Gene Expression in <i>Arabidopsis thaliana</i> Using an Excised Single Leaf.","authors":"Kohei Kitawaki, Ryota Mihara, Yausko Ito-Inaba, Takehito Inaba","doi":"10.21769/BioProtoc.5319","DOIUrl":"https://doi.org/10.21769/BioProtoc.5319","url":null,"abstract":"<p><p>The identification of chemical compounds that affect intracellular processes has greatly contributed to the understanding of developmental regulation in plants. In this protocol, we describe a method for identifying chemical compounds that affect cold-regulated gene expression in <i>Arabidopsis thaliana</i>. Specifically, we generated <i>Arabidopsis</i> plants harboring a COLD-REGULATED 15A <i>(COR15A)</i> promoter::luciferase (<i>COR15Apro::LUC</i>) construct and grew them in a submerged liquid culture. Using a single true leaf excised from <i>COR15Apro::LUC</i> plants and 96-well culture plates, we performed high-throughput screening of chemical compounds that inhibit cold-induction of <i>COR15Apro::LUC</i>. Luciferase activity was detected using a microplate reader and a chemiluminescence imaging device. This protocol can be easily used for the identification of chemical compounds that regulate other processes, being versatile with respect to equipment. Key features • High-throughput screening of chemical compounds that affect cold-regulated gene expression is possible using a single leaf excised from <i>Arabidopsis</i> grown in a submerged culture. • Screening is based on luciferase activity derived from an excised single leaf. • Direct measurement of luciferase activity is possible using a microplate reader and a chemiluminescence imaging device. • This protocol can be easily used for the identification of chemical compounds that regulate other processes.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 10","pages":"e5319"},"PeriodicalIF":1.0,"publicationDate":"2025-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12104835/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144164098","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"I-PREFR: Inverse PCR-Based Restriction Enzyme FRee Unidirectional Strategy for Rapid Markerless Chromosomal Gene Deletion and Reconstitution in Bacteria Using Suicide Vectors.","authors":"Rekha Rana, Anushika Sharma, Ashish Dutta, Prabhu B Patil","doi":"10.21769/BioProtoc.5314","DOIUrl":"https://doi.org/10.21769/BioProtoc.5314","url":null,"abstract":"<p><p>The standard protocols for allelic exchange using homologous recombination deploy suicide vectors with negative selection markers. However, the use of multiple restriction enzymes to generate sticky ends in the vector and the insert for cloning is time-consuming, resource-intensive, and challenging. The advent of next-generation proofreading enzymes is enabling researchers to routinely carry out long-range PCR. Hence, amplifying 5-6 kb of complete low-complex DNA cloning vectors and 2-3 kb of complex genomic regions is much easier. Here, we report a simple, accurate, rapid, and unidirectional approach for chromosomal in-frame gene deletion and complementation by reconstitution of the full-length gene without using any restriction enzymes. The method requires long-range PCR using Phusion polymerase to linearize the vector and amplify the target gene to create a recombinant vector (pRM1) and further inverse PCR amplification of pRM1 to create a recombinant vector (pRM4) with a deleted version of the gene. The cloning steps involve the use of kinase and ligase for phosphorylation and ligation steps, respectively. The recombinant plasmid, pRM4, is finally transformed into electrocompetent cells of <i>Xanthomonas sontii</i>, a gram-negative phytobacterium, for final genomic integration/excision to obtain an in-frame gene deletion mutant (PPL1RM15). Gene reconstitution for complementation is carried out by electroporating the deletion mutant with the recombinant plasmid (pRM1) carrying the wild-type allele. Clean gene mutation, allele restoration, and plasmid excision are confirmed using whole-genome sequencing. Key features • The protocol is cost-effective and simple, eliminating the need for restriction enzymes and multiple sets of lengthy primers with restriction sites. • The protocol requires only kinase, ligase, and polymerase, along with three sets of standard-sized desalted primers. • The protocol is unidirectional; no need to create two different recombinant plasmids for gene deletion and complementation by reconstitution of the full-length gene. • The difference in size between empty and recombinant vectors facilitates the easy screening of transformed <i>Escherichia coli</i> colonies through colony PCR. • The strategy can be applied to any bacteria using a suitable suicide vector with appropriate positive and negative selection markers.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 10","pages":"e5314"},"PeriodicalIF":1.0,"publicationDate":"2025-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12104833/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144164101","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Development of Polyethylene Glycol Diacrylate-Based Micropattern Substrate to Study the Interplay Between Surface Topography and Cellular Response for Tissue Engineering Applications.","authors":"Mohd Khairul Akma Darwis, Victoria Levario-Diaz, Sadaf Pashapour, Jonah Luka Voigt, Eloïse Lebaudy, Norhayati Sabani, Ahmad Shuhaimi Abu Bakar, Nihal Engin Vrana, Philippe Lavalle, Elisabetta Ada Cavalcanti-Adam, Siti Hawa Ngalim","doi":"10.21769/BioProtoc.5323","DOIUrl":"https://doi.org/10.21769/BioProtoc.5323","url":null,"abstract":"<p><p>A key goal in the bioengineering field is the development of surface patterning of proteins that guide and control cellular organization. To this end, we developed a method to create a microstructured hydrogel based on soft-lithography techniques using polydimethylsiloxane (PDMS) and polyethylene glycol diacrylate (PEGDA). This approach involves the design of microfluidic geometries using graphical software, employing PDMS as a mold and leaving PEGDA as a substrate for the fabrication of microstructures and, thus, patterning extracellular matrix (ECM) proteins to promote cell adhesion. The combination of these techniques allows the fabrication of hydrogel microstructures without following conventional photolithography methods, such as the use of a photomask, the alignment required to produce the patterns, and the associated expenses. This study highlights the versatility and potential of PEGDA-based hydrogels as platforms to advance tissue engineering strategies. Key features • This protocol focuses on investigating the feasibility of patterning PEGDA as a substrate for protein surface patterning and further tissue engineering applications. • Optimization of the fabrication of PEGDA hydrogels into simple shapes and angular patterns, ensuring a robust substrate capable of guiding cellular responses.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 10","pages":"e5323"},"PeriodicalIF":1.0,"publicationDate":"2025-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12104878/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144164039","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Quantifying Thrombogenicity: A Bioanalytical Protocol for the Absorbance-Based Assessment of Vascular Implants with Plasma.","authors":"Anna Sallee, Aurora Battistella, Richard Johnson, Alexander Duong, Wei Tan, Novella M Keeling","doi":"10.21769/BioProtoc.5316","DOIUrl":"https://doi.org/10.21769/BioProtoc.5316","url":null,"abstract":"<p><p>Assessing thrombogenicity is crucial for evaluating biomaterials, especially those that interface with flowing blood, such as cardiovascular implants, including vascular stents, grafts, and stent-grafts. To standardize thrombogenicity assessments, we use human plasma and quantify the light absorbance associated with the biomaterial. For this evaluation, various tubular vascular implants from leading brands-such as bare-metal stents, drug-eluting stents, vascular grafts, and stent-grafts-are longitudinally sectioned into small pieces and placed in a low-adhesion 96-well plate. Using either platelet-rich plasma (PRP) or platelet-poor plasma (PPP), we measure the absorbance of light passing through the plate over an hour and plot the resulting curve. This method quantifies the thrombogenicity of a biomaterial under controlled conditions. Key factors examined include anticoagulants, platelet presence, and surface-coating molecules to assess their impact on thrombogenicity. Using this simple, reproducible protocol, we demonstrated (a) the relative efficacy of various anticoagulants in thrombogenicity assessments, and (b) the effectiveness of vascular coating molecules in reducing thrombogenicity on stents. This streamlined approach offers valuable insights for optimizing biomaterial performance in vascular implants. Unlike conventional clotting assays, which focus on standardized blood clotting mechanisms, this assay is tailored to evaluate biomaterials and external parameters influencing thrombogenicity. Key features • This protocol is a static, in vitro, quantitative assessment of thrombogenicity for both novel and commercial vascular biomaterials with flat or tubular geometries. • Thrombogenicity is assessed by evaluating the opaqueness of platelet-rich or platelet-poor plasma over time to measure fibrin formation. • Various anticoagulants such as sodium citrate, sodium ethylenediaminetetraacetic acid, and sodium heparin are compared to evaluate the efficacy of the protocol. • This protocol compares the thrombogenicity of various commercial stents, grafts, and stent-graft hybrids, as well as novel molecular coatings.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 10","pages":"e5316"},"PeriodicalIF":1.0,"publicationDate":"2025-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12104834/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144164049","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Stable ¹³C-glutamine Tracing Resolved Metabolomics for Cancer Metabolism Study.","authors":"Yaogang Zhong, Liqing He, Xinmin Yin, Logan Mazik, Xiang Zhang, Deliang Guo","doi":"10.21769/BioProtoc.5322","DOIUrl":"https://doi.org/10.21769/BioProtoc.5322","url":null,"abstract":"<p><p>Stable isotopes have frequently been used to study metabolic processes in live cells both in vitro and in vivo. Glutamine, the most abundant amino acid in human blood, plays multiple roles in cellular metabolism by contributing to the production of nucleotides, lipids, glutathione, and other amino acids. It also supports energy production via anaplerosis of tricarboxylic acid cycle intermediates. While ¹³C-glutamine has been extensively employed to study glutamine metabolism in various cell types, detailed analyses of specific lipids derived from ¹³C-glutamine via the reductive carboxylation pathway are limited. In this protocol, we present a detailed procedure to investigate glutamine metabolism in human glioblastoma (GBM) cells by conducting ¹³C-glutamine tracing coupled with untargeted metabolomics analysis using liquid chromatography-mass spectrometry (LC-MS/MS). The method includes step-by-step instructions for the extraction and detection of polar metabolites and long-chain fatty acids (LCFAs) derived from ¹³C-glutamine in GBM cells. Notably, this approach enables the distinction between isomers of two monounsaturated FAs with identical masses: palmitoleic acid (16:1n-7) (cis-9-hexadecenoic acid) and palmitelaidic acid (16:1n-7) (trans-9-hexadecenoic acid) derived from ¹³C-glutamine through the reductive carboxylation process. In addition, using this protocol, we also unveil previously unknown metabolic alterations in GBM cells following lysosome inhibition by the antipsychotic drug pimozide. Key features • Methods for analyzing the flux of the stable isotope ¹³C-glutamine in cancer cells and identifying its derived polar metabolites and long-chain fatty acids (LCFAs). • Distinguishes isomers of long-chain fatty acids, such as palmitoleic acid (16:1n-7) (cis-9-Hexadecenoic acid) and palmitelaidic acid (16:1n-7) (trans-9-Hexadecenoic acid), which share the exact same mass. • The method is utilized to investigate glutamine metabolism reprogramming in cancer cells following lysosome inhibition.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 10","pages":"e5322"},"PeriodicalIF":1.0,"publicationDate":"2025-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12104877/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144164283","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Egg Microinjection for the Silkworm <i>Bombyx mori</i>.","authors":"Hayato Yamada, Toshinobu Yaginuma, Keisuke Mase, Teruyuki Niimi","doi":"10.21769/BioProtoc.5317","DOIUrl":"https://doi.org/10.21769/BioProtoc.5317","url":null,"abstract":"<p><p>The silkworm <i>Bombyx mori</i> has been extensively utilized in sericulture and serves as a representative model insect of Lepidoptera in various fields of life sciences and applied research. In recent years, its significance has further increased in molecular genetics and functional genomics. Germline transformation and genome editing in <i>B. mori</i> require the injection of vector solutions into early embryos; however, the thick eggshell of <i>B. mori</i> presents a significant challenge for microinjection. Conventional methods involve arranging eggs, pre-pierced with a tungsten needle, followed by solution injection, making the process both time-consuming and technically demanding. Here, we describe a simplified and more efficient microinjection protocol. Unlike conventional approaches, our method eliminates the need for egg removal from the egg-laying sheet and egg alignment on the slide glass by allowing injections to be performed directly on eggs retained on the egg-laying sheet. A thick-walled glass capillary, capable of penetrating the rigid eggshell, is used to directly pierce the eggshell and deliver the solution. By eliminating the need for egg alignment and micromanipulator operation, this protocol significantly enhances efficiency, enabling higher-throughput embryo injections within a shorter time frame. Moreover, this approach holds potential for application to other insect species with similarly thick eggshells. Key features • A simple and efficient microinjection method for silkworm eggs. • Eliminates the need for egg alignment by injecting directly into eggs attached to the oviposition substrate. • Conducted without a micromanipulator or tungsten needle to pierce the eggshell, using only a handheld thick-walled glass capillary. • Applicable to other insect species with thick eggshells.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 10","pages":"e5317"},"PeriodicalIF":1.0,"publicationDate":"2025-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12104832/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144164005","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"RNA PolyA Tailing Assay to Qualitatively Analyze Circular RNA Manufacturing.","authors":"Zih-Shiuan Chuang, Chan-I Su, Hsin-I Wang, Chia-Yi Yu","doi":"10.21769/BioProtoc.5310","DOIUrl":"https://doi.org/10.21769/BioProtoc.5310","url":null,"abstract":"<p><p>A covalently closed loop structure provides circular RNA (circRNA) with more stability than conventional RNAs in linear form, making circRNA an emerging tool in RNA therapeutics. The qualification and quantification of circRNA after production is critical for its design and effectiveness assessments, particularly when the following applications could be affected by byproduct RNAs. Despite PCR-based methods effectively detecting low-abundance circRNA, they are unsuitable for assessing uncircularized RNA in a mass production fraction to maintain quality control. Here, we present a straightforward protocol for evaluating uncircularized byproduct RNAs from circRNA production. This method enrolls the template-independent RNA polymerase activity to add adenine tails (polyA) to the 3' ends of a linear RNA, making it easy to distinguish trace byproducts or uncircularized RNA from a pool of mass circRNA products. With conventional linear RNA and RNase R-treated circRNA as the positive and negative controls, the purity of a circRNA preparation could be readily resolved. Regardless of circRNA production strategies, this protocol provides a reliable and practical way to ensure the consistent quality of homemade circRNAs or to recheck circRNA quality from commercial manufacturing. Key features • The RNA circularization assessment relies on detecting a free 3' end OH group of the non-circRNA in the circRNA products. • An RNA denaturing electrophoresis is required to detect nucleotide length changes of the non-circRNA post-3' end tailing. • Rather than quantitatively, the molecular weight changes qualitatively highlight the trace non-circRNA in a circRNA preparation. • While there are different techniques for producing circRNA, the tailing method is effective for most, detecting leftover linear RNA contaminants.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 10","pages":"e5310"},"PeriodicalIF":1.0,"publicationDate":"2025-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12104830/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144164166","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"From Bedside to Desktop: A Data Protocol for Normative Intracranial EEG and Abnormality Mapping.","authors":"Heather Woodhouse, Sarah J Gascoigne, Gerard Hall, Callum Simpson, Nathan Evans, Gabrielle M Schroeder, Peter N Taylor, Yujiang Wang","doi":"10.21769/BioProtoc.5321","DOIUrl":"https://doi.org/10.21769/BioProtoc.5321","url":null,"abstract":"<p><p>Normative mapping is a framework used to map population-level features of health-related variables. It is widely used in neuroscience research, but the literature lacks established protocols in modalities that do not support healthy control measurements, such as intracranial electroencephalograms (icEEG). An icEEG normative map would allow researchers to learn about population-level brain activity and enable the comparison of individual data against these norms to identify abnormalities. Currently, no standardised guide exists for transforming clinical data into a normative, regional icEEG map. Papers often cite different software and numerous articles to summarise the lengthy method, making it laborious for other researchers to understand or apply the process. Our protocol seeks to fill this gap by providing a dataflow guide and key decision points that summarise existing methods. This protocol was heavily used in published works from our own lab (twelve peer-reviewed journal publications). Briefly, we take as input the icEEG recordings and neuroimaging data from people with epilepsy who are undergoing evaluation for resective surgery. As final outputs, we obtain a normative icEEG map, comprising signal properties localised to brain regions. Optionally, we can also process new subjects through the same pipeline and obtain their z-scores (or centiles) in each brain region for abnormality detection and localisation. To date, a single, cohesive dataflow pipeline for generating normative icEEG maps, along with abnormality mapping, has not been created. We envisage that this dataflow guide will not only increase understanding and application of normative mapping methods but will also improve the consistency and quality of studies in the field. Key features • Resultant normative maps can be used to test a broad range of hypotheses in the neuroscience field. • Provides a more detailed walkthrough of the methods in the normative mapping study conducted by Taylor et al. [1] and other related publications [2-12]. • Offers flexibility: readers can tailor the final output by considering key decision points included throughout the protocol. • Involves sub-pipelines, which may be useful to researchers in isolation (i.e., icEEG electrode localisation and/or interictal segment selection).</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 10","pages":"e5321"},"PeriodicalIF":1.0,"publicationDate":"2025-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12104836/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144164095","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"<i>Mycobacterium smegmatis</i> Ribosome Purification, Co-sedimentation, and Subunit Association Assay.","authors":"Aneek Banerjee, Sandip Dey, Krishnamoorthi Srinivasan, Ankit Dhur, Priya Baid, Jayati Sengupta","doi":"10.21769/BioProtoc.5318","DOIUrl":"https://doi.org/10.21769/BioProtoc.5318","url":null,"abstract":"<p><p>The ribosome, a complex macromolecular machine, plays a vital role in cellular translation. To investigate its structure and conduct in vitro experiments, isolating the ribosomes from cells is the first step. While isolating ribosomes from bacterial cells is routine, obtaining them from mycobacteria proves challenging due to the protective mycolic acid layer, which hinders cell lysis. In this study, we present a straightforward and efficient protocol for isolating ribosomes from <i>Mycobacterium smegmatis</i>. Additionally, we introduce a co-sedimentation assay using density gradient ultracentrifugation, providing a simple yet powerful method for studying ribosome-protein interactions. The re-association assay also offers a practical approach for obtaining tRNA-free 70S ribosomes and evaluating the anti-association properties of potential ligands. While these assays are commonly used, our protocol stands out for its simplicity, requiring limited specialized instruments. These methods can also be scaled up or down per requirement. By employing sonication for cell rupture and utilizing basic lab equipment for ultracentrifugation-based assays, our method greatly simplifies ribosome isolation and related research. Key features • Cellular ribosome isolation from <i>Mycobacterium smegmatis</i> using an optimized sonication cycle. • Detection of ribosome-protein interactions through density gradient ultracentrifugation. • Simple steps to obtain tRNA-free 70S ribosomes that are crucial for several biochemical experiments. • Screening of proteins or ligands for their ribosomal anti-association activity.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 10","pages":"e5318"},"PeriodicalIF":1.0,"publicationDate":"2025-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12117347/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144175681","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"PBMC-Humanized Mouse Model for Multiple Sclerosis: Studying Immune Changes and CNS Involvement.","authors":"Anastasia Dagkonaki, Irini Papazian, Vasileios Gouzouasis, Ariadni Karles, Maria Kourouvani, Theodore Tselios, Eirini Fragkiadaki, Lesley Probert","doi":"10.21769/BioProtoc.5312","DOIUrl":"https://doi.org/10.21769/BioProtoc.5312","url":null,"abstract":"<p><p>Humanized immune system (HIS) mice are powerful tools for studying human immune system function and dysfunction and developing human-specific immunotherapeutics. The availability of sophisticated super immunodeficient mouse strains has allowed immune system humanization using transplants of human peripheral blood mononuclear cells (PBMC) or hematopoietic stem cells. HIS mice are used extensively in immune-oncology, while there are fewer studies in autoimmunity, especially multiple sclerosis (MS). Using the protocol described here, we generated HIS mice that show key features of MS not represented in other widely used MS models [1]. Severely immunodeficient NOD.Cg-<i>B2m^em1Tac Prkdc^scid Il2rg^tm1Sug</i> /JicTac (B2m-NOG) mice, which lack murine B, T, and NK cells and murine major histocompatibility class I molecules and have defective innate immune responses, were transplanted with PBMC from HLA-DRB1-genotyped MS patients and healthy donors. Mice were successfully engrafted with hCD4 and hCD8 T and B lymphocytes and developed both spontaneous and experimental autoimmune encephalomyelitis (EAE)-enhanced T-cell lesions in the central nervous system. B-cell engraftment was highest in mice receiving cells from MS patients with serological evidence for Epstein-Barr virus (EBV) reactivation. This humanized MS model shows advantages over EAE, particularly spontaneous hCD8 T-cell lesions in the brain and spinal cord, mixed hCD8/hCD4 T-cell lesions in EAE-immunized mice, and more severe lesions in mice engrafted with PBMC from MS donors carrying the DRB1*15 MS susceptibility allele compared to DRB1*15-positive healthy and DRB1*13-positive MS donors. MS HIS mice represent simple and rapid tools for investigating human immunopathology and the efficacy of therapeutics at a personalized level. Key features • Humanization of severely immunodeficient B2m-NOG mice with human PBMC. • Engraftment analysis of human immune system in mice using multicolor flow cytometry. • Animal familiarization and handling techniques. • Epstein-Barr virus latency evaluation in human plasma. • Ex vivo characterization of engrafted T-cell cytokine responses.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 10","pages":"e5312"},"PeriodicalIF":1.0,"publicationDate":"2025-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12104831/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144164104","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}