Current protocols最新文献

{"title":"Synthesis of Oligodeoxynucleotide Containing Pseudo-Deoxycytidine and Its Triphosphate Derivative","authors":"Ryo Miyahara,&nbsp;Yosuke Taniguchi","doi":"10.1002/cpz1.70101","DOIUrl":"https://doi.org/10.1002/cpz1.70101","url":null,"abstract":"<p>This article describes a detailed synthetic protocol for the preparation of oligodeoxynucleotide (ODN) containing pseudo-deoxycytidine (ψdC) and its triphosphate derivative (ψdCTP). These molecules were synthesized as novel compounds that recognize iso-2'-deoxyguanosine (iso-dG) in DNA. Iso-dG is one of the tautomers of 2-hydroxy-2'-deoxyadenosine (2-OH-dA), which is known as an oxidatively damaged nucleobase, and its selective recognition in DNA is expected to play a very important role in the diagnosis and pathogenesis of diseases. The hydroxyl groups of the known glycal compound were protected with silyl groups, and then coupled with 5-iodouracil under Mizorogi-Heck reaction conditions, yielding ψdU after desilylation and diastereoselective reduction. The endocyclic amino group of ψdU was protected by the benzyl group. Subsequently, the carbonyl group at the 6-position of the nucleobase was activated and converted to an amino group through treatment with aqueous ammonia. The benzyl group was removed, and the exocyclic amino group was protected with a benzoyl group. On one hand, the silyl groups at the 3’ and 5’ positions were deprotected, converted into a phosphoramidite unit, and incorporated into an ODN. On the other hand, the hydroxyl group at the 5’ position was selectively deprotected and then directly converted into the triphosphate using Van Boom's reagent under acidic conditions. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Synthesis of ODNs having ψdC and ψdCTP</p><p><b>Basic Protocol 2</b>: Melting temperature of duplex formation between ODNs containing ψdC unit and 2-OH-dA</p><p><b>Basic Protocol 3</b>: A single nucleotide primer extension reaction of ψdCTP for a template strand containing 2-OH-dA</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 3","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-03-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70101","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143632700","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":"Expression and Purification of SARS-related Spike Glycoproteins for Cryo-EM Analysis","authors":"Francesca R. Hills,&nbsp;Fátima Jorge,&nbsp;Laura N. Burga,&nbsp;Mihnea Bostina","doi":"10.1002/cpz1.70115","DOIUrl":"https://doi.org/10.1002/cpz1.70115","url":null,"abstract":"<p><i>Coronaviridae</i> spike glycoproteins mediate viral entry and fusion to host cells through binding to host receptors (i.e., ACE2, DPP4) and are key components in determining viral host range, making them targets for antiviral research. Here, we describe the expression, purification, and characterization of recombinant spike proteins to aid in protein characterization and analysis. These protocols were used for the production of spike glycoproteins from civet, pangolin, and bat coronaviruses, as well as high-resolution cryo-electron microscopy (cryo-EM) structural analysis of bat and civet host coronavirus spike glycoproteins (Hills et al., 2024). © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Expression and purification of SARS-CoV spike protein from ExpiCHO cells</p><p><b>Basic Protocol 2</b>: Preparation of SARS-CoV spike protein for visualization by negative-stain transmission electron microscopy and cryo-electron microscopy</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 3","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-03-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70115","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143571348","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":"A Physiologically Relevant In Vitro Model of Nonreplicating Persistent Mycobacterium tuberculosis in Caseum","authors":"Min Xie,&nbsp;Paulina Osiecki,&nbsp;Suyapa Rodriguez,&nbsp;Véronique Dartois,&nbsp;Jansy Sarathy","doi":"10.1002/cpz1.70118","DOIUrl":"https://doi.org/10.1002/cpz1.70118","url":null,"abstract":"<p>Tuberculosis (TB) remains one of the leading infectious causes of death worldwide. Persistent bacterial populations in specific microenvironments within the host hamper efficient TB chemotherapy. Caseum in the necrotic core of closed granulomas and cavities of pulmonary TB patients can harbor high burdens of drug-tolerant <i>Mycobacterium tuberculosis</i> (MTB) bacilli, making them particularly difficult to sterilize. Here, we describe protocols for the generation of a surrogate matrix using lipid-rich macrophages to mimic the unique composition of caseum <i>in vivo</i>. Importantly, this caseum surrogate induces metabolic and physiological changes within MTB that reproduce the nonreplicating drug-tolerant phenotype of the pathogen in the native caseous environment, making it advantageous over alternative <i>in vitro</i> models of nonreplicating persistent (NRP) MTB. The protocols include culture of THP-1 monocytes, stimulation of lipid droplet accumulation, lysis and denaturation of the foamy macrophages, inoculation and preadaptation of MTB bacilli in the caseum surrogate, and evaluation of drug bactericidal activity against the NRP population. This novel <i>in vitro</i> model is being used to screen for potent bactericidal antimicrobial agents and to identify vulnerable drug targets, among a variety of other applications, thereby reducing our reliance on <i>in vivo</i> models. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Caseum surrogate preparation from γ-irradiated <i>M. tuberculosis</i>–induced foamy THP-1 monocyte–derived macrophages (THPMs)</p><p><b>Alternate Protocol 1</b>: Caseum surrogate preparation from stearic acid–induced THPMs</p><p><b>Basic Protocol 2</b>: Generation of nonreplicating persistent <i>M. tuberculosis</i> and drug susceptibility testing</p><p><b>Alternate Protocol 2</b>: Higher-throughput drug susceptibility screening using caseum surrogate</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 3","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-03-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70118","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143571349","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":"Optimized, Efficient Measurement of the Expression of Undifferentiated Stem Cell Markers in Human Induced Pluripotent Stem Cells (iPSCs) by Flow Cytometry","authors":"Vaishanavi Saware,&nbsp;Wendy Runyon,&nbsp;Sam Hu,&nbsp;Benjamin van Soldt,&nbsp;Ritu Kumar,&nbsp;Jane Srivastava","doi":"10.1002/cpz1.70105","DOIUrl":"https://doi.org/10.1002/cpz1.70105","url":null,"abstract":"<p>Induced pluripotent stem cells (iPSCs) have revolutionized the fields of regenerative medicine, disease modeling, and drug discovery. However, the usage of iPSCs for various applications has been hampered by the observed line-to-line variability in their differentiation capacity. Therefore, it is important to verify the pluripotent status of iPSCs. A very effective way to define the pluripotent state of iPSCs is by evaluating the expression of established undifferentiated stem cell markers. A bona fide iPSC must have high, homogeneous expression of these markers. Here, we present a cost-effective platform that can be readily utilized by researchers to define the pluripotency status of iPSCs by measuring the expression of surface and intracellular markers by flow cytometry. © 2025 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: iPSC culture and collection for flow cytometry analysis</p><p><b>Basic Protocol 2</b>: Staining of iPSCs for extracellular and intracellular undifferentiated stem cell markers</p><p><b>Basic Protocol 3</b>: Flow cytometry acquisition</p><p><b>Basic Protocol 4</b>: Flow cytometry data analysis</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 3","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-03-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143530157","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Development, Characterization, and Therapeutic Utility of Paclitaxel-Resistant Breast and Gastric Cancer In Vitro and In Vivo Models","authors":"Feng Tang,&nbsp;Hong Xu,&nbsp;Wen Cui,&nbsp;Xuelin Wang,&nbsp;Fangyi Ding,&nbsp;Yihan Zhang,&nbsp;Xiangnan Qiang,&nbsp;Qingyang Gu,&nbsp;Dong Wang,&nbsp;Zhixiang Zhang","doi":"10.1002/cpz1.70113","DOIUrl":"https://doi.org/10.1002/cpz1.70113","url":null,"abstract":"<p>Paclitaxel, one of the most commonly used anticancer agents, is employed in the treatment of a range of malignant tumors. However, resistance is one of the major barriers to successful therapy. Despite its clinical relevance, the molecular mechanisms underlying paclitaxel resistance remain poorly understood. In this protocol, we describe the methods for establishing paclitaxel-resistant tumor models both in vitro and in vivo, and how we investigated the underlying mechanisms of resistance. Additionally, we evaluated the potential of combination therapies to overcome paclitaxel resistance in these models. © 2025 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Generation of paclitaxel-resistant breast cancer model in vivo.</p><p><b>Basic Protocol 2</b>: Generation of paclitaxel-resistant gastric cancer model in vitro.</p><p><b>Basic Protocol 3</b>: Validation of drug resistance in vivo.</p><p><b>Basic Protocol 4</b>: In vitro and in vivo evaluation of combination therapy in paclitaxel-resistant models.</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 3","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-03-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143534038","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Imaging the Intestinal Transcriptome With Multiplexed Error-Robust Fluorescence In Situ Hybridization (MERFISH)","authors":"Paolo Cadinu,&nbsp;Evan Yang,&nbsp;Rosalind J. Xu,&nbsp;Brianna R. Watson,&nbsp;Josh Luce,&nbsp;Jeffrey R. Moffitt","doi":"10.1002/cpz1.70111","DOIUrl":"https://doi.org/10.1002/cpz1.70111","url":null,"abstract":"<p>Multiplexed error-robust fluorescence in situ hybridization (MERFISH) is a massively multiplexed single RNA–molecule imaging technique capable of spatially resolved single-cell transcriptomic profiling of thousands of genes in millions of cells within intact tissue slices. Initially introduced for brain tissues, MERFISH has since been extended to other tissues, where rapid RNA degradation during the preparation process can pose challenges. This protocol outlines the application of MERFISH in one such challenging tissue, the mammalian gastrointestinal tract. We describe two complementary protocols leveraging either fresh frozen or fixed frozen approaches and describe methods for combining RNA imaging with immunofluorescence. While these protocols were designed and validated in gut tissues, we anticipate that they will be useful resources for the application to other challenging tissue types. © 2025 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Fixed-frozen sample preparation</p><p><b>Basic Protocol 2</b>: Fresh-frozen sample preparation</p><p><b>Basic Protocol 3</b>: Encoding probe construction</p><p><b>Basic Protocol 4</b>: MERFISH imaging</p><p><b>Basic Protocol 5</b>: Image decoding</p><p><b>Support Protocol 1</b>: Coverslip silanization</p><p><b>Support Protocol 2</b>: Poly-<span>d</span>-lysine (PDL) coating of the coverslips</p><p><b>Support Protocol 3</b>: Hybridization buffer preparation</p><p><b>Support Protocol 4</b>: Trolox quinone stock preparation</p><p><b>Support Protocol 5</b>: TCEP stock preparation</p><p><b>Alternate Protocol 1</b>: MERFISH-compatible immunofluorescent boundary stains in fresh frozen tissue</p><p><b>Alternate Protocol 2</b>: Immunofluorescent boundary stains with methacrylate-NHS-anchored antibodies for PFA-fixed samples</p><p><b>Alternate Protocol 3</b>: Guanidine-HCl tissue clearing</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 3","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-03-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143535951","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Protocol for Controlling the Strand Selectivity of siRNA Using Acyclic Artificial Nucleic Acids.","authors":"Jumpei Ariyoshi, Hiroyuki Asanuma, Yukiko Kamiya","doi":"10.1002/cpz1.70103","DOIUrl":"https://doi.org/10.1002/cpz1.70103","url":null,"abstract":"<p><p>Small interfering RNA (siRNA) has emerged as a promising therapeutic candidate against previously intractable diseases. An effective siRNA must have high on-target activity while off-target effects are minimized. This balance can be achieved by enhancing the selectivity of the antisense strand through sequence optimization and appropriate chemical modifications. Acyclic artificial nucleic acids such as serinol nucleic acids (SNA) have demonstrated on-target activity while suppressing off-target effects. This article provides guidelines for designing SNA-modified siRNA and outlines a method for the experimental evaluation of the on-target and off-target activities of siRNAs, ensuring accurate functional validation in cell systems. These protocols benefit researchers developing siRNA-based therapeutics to optimize siRNA selectivity and efficacy while minimizing off-target effects through innovative design strategies. © 2025 Wiley Periodicals LLC. Basic Protocol 1: Design of SNA-modified siRNA Basic Protocol 2: Design and preparation of vector plasmids using inverse PCR Alternate Protocol: Design and preparation of vector plasmid using restriction enzymes and ligase Basic Protocol 3: Evaluation of the on- and off-target effects of siRNAs using the dual-luciferase assay Support Protocol 1: Agarose gel electrophoresis and protocol for purifying DNA from gels Support Protocol 2: Transformation and amplification of plasmids.</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 3","pages":"e70103"},"PeriodicalIF":0.0,"publicationDate":"2025-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143664892","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Visualization of Efavirenz-Induced Lipid Alterations in the Mouse Brain Using MALDI Mass Spectrometry Imaging","authors":"Nav Raj Phulara,&nbsp;Herana Kamal Seneviratne","doi":"10.1002/cpz1.70108","DOIUrl":"https://doi.org/10.1002/cpz1.70108","url":null,"abstract":"<p>This article highlights experimental procedures and troubleshooting tips for the utilization of matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) methods for detecting and visualizing lipid alterations in the mouse brain tissue in response to efavirenz (EFV) treatment. To investigate drug-induced adverse effects, it is becoming increasingly important to understand the spatial alterations of lipid molecules in the target organs. EFV is a non-nucleoside reverse transcriptase inhibitor commonly used for HIV treatment in combination with other antiretrovirals. Importantly, EFV is a drug that is included in the World Health Organization's list of essential medications. However, EFV is known to be associated with neurotoxicity. To date, the mechanisms underlying EFV-induced neurotoxicity have not been fully elucidated. Therefore, it is important to gain understanding of the effect of EFV on the brain. It is known that the brain is composed of different neuroanatomical regions that are abundant in lipids. Described here is the use of a chemical imaging strategy, MALDI MSI, to detect, identify, and visualize the spatial localization of several lipid species across the brain tissue sections along with their alterations in response to EFV treatment. The set of protocols consists of three major parts: lipid detection, identification, and tissue imaging. Lipid detection includes testing different chemical matrices and how they facilitate the detection of analytes, which is then followed by identification. Collision-induced dissociation is employed to verify the identity of the lipid molecules. Lastly, tissue imaging experiments are performed to generate the spatial localization profiles of the lipids. The protocols described in this article can be employed to spatially visualize alterations in the lipid molecules in response to drug treatment. © 2025 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: MALDI mass spectrometry (MALDI MS) profiling experiments for detection of lipids</p><p><b>Basic Protocol 2</b>: MALDI MS imaging of lipid molecules in mouse brain tissues</p><p><b>Basic Protocol 3</b>: MALDI MS data processing and analysis</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 2","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-02-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143490037","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Engineering Base Changes and Epitope-Tagged Alleles in Mice Using Cas9 RNA-Guided Nuclease","authors":"Marina Gertsenstein,&nbsp;Lauri G. Lintott,&nbsp;Lauryl M. J. Nutter","doi":"10.1002/cpz1.70109","DOIUrl":"https://doi.org/10.1002/cpz1.70109","url":null,"abstract":"<p>Mice carrying patient-associated base changes are powerful tools to define the causality of single-nucleotide variants to disease states. Epitope tags enable immuno-based studies of genes for which no antibodies are available. These alleles enable detailed and precise developmental, mechanistic, and translational research. The first step in generating these alleles is to identify within the target sequence—the orthologous sequence for base changes or the N or C terminus for epitope tags—appropriate Cas9 protospacer sequences. Subsequent steps include design and acquisition of a single-stranded oligonucleotide repair template, synthesis of a single guide RNA (sgRNA), collection of zygotes, and microinjection or electroporation of zygotes with Cas9 mRNA or protein, sgRNA, and repair template followed by screening born mice for the presence of the desired sequence change. Quality control of mouse lines includes screening for random or multicopy insertions of the repair template and, depending on sgRNA sequence, off-target sequence variation introduced by Cas9. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Single guide RNA design and synthesis</p><p><b>Alternate Protocol 1</b>: Single guide RNA synthesis by primer extension and in vitro transcription</p><p><b>Basic Protocol 2</b>: Design of oligonucleotide repair template</p><p><b>Basic Protocol 3</b>: Preparation of RNA mixture for microinjection</p><p><b>Support Protocol 1</b>: Preparation of microinjection buffer</p><p><b>Alternate Protocol 2</b>: Preparation of RNP complexes for electroporation</p><p><b>Basic Protocol 4</b>: Collection and preparation of mouse zygotes for microinjection or electroporation</p><p><b>Basic Protocol 5</b>: Electroporation of Cas9 RNP into zygotes using cuvettes</p><p><b>Alternate Protocol 3</b>: Electroporation of Cas9 RNP into zygotes using electrode slides</p><p><b>Basic Protocol 6</b>: Screening and quality control of derived mice</p><p><b>Support Protocol 2</b>: Deconvoluting multiple sequence chromatograms with DECODR</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 2","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-02-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70109","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143489743","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 Syngeneic Murine Glioma Models with Somatic Mismatch Repair Deficiency to Study Therapeutic Responses to Alkylating Agents and Immunotherapy","authors":"Deepti Bhatt,&nbsp;Ranjini K. Sundaram,&nbsp;Karla S. Lugo López,&nbsp;Teresa Lee,&nbsp;Susan E. Gueble,&nbsp;Juan C. Vasquez","doi":"10.1002/cpz1.70097","DOIUrl":"https://doi.org/10.1002/cpz1.70097","url":null,"abstract":"<p>Glioblastoma (GBM) carries a dismal prognosis, with a median survival of less than 15 months. Temozolomide (TMZ), the standard frontline chemotherapeutic for GBM, is an alkylating agent that generates DNA <i>O</i>⁶-methylguanine (O⁶MeG) lesions. Without O⁶MeG-methyltransferase (MGMT), this lesion triggers the mismatch repair (MMR) pathway and leads to cytotoxicity via futile cycling. TMZ resistance frequently arises via the somatic acquisition of MMR deficiency (MMRd). Moreover, DNA-damaging agents have been shown capable of increasing tumor immunogenicity and improving response to immune checkpoint blockade (ICB), which has had limited success in glioma. The study of how alkylating chemotherapy such as TMZ impacts antitumor immunity in glioma has been hindered by a lack of immunocompetent models that incorporate relevant DNA repair genotypes. Here, we used CRISPR/Cas9 to generate models isogenic for knockout (KO) of Mlh1 in the syngeneic SB28 murine glioma cell line. MMR KO models readily formed intracranial tumors and exhibited <i>in vitro</i> and <i>in vivo</i> resistance to TMZ. In contrast, MMR KO cells maintained sensitivity to KL-50, a newly developed alkylating compound that exerts MGMT-dependent, MMR-independent cytotoxicity. Lastly, MMR KO tumors remained resistant to ICB, mirroring the lack of response seen in patients with somatic MMRd GBM. The development of syngeneic, immunologically cold glioma models with somatic loss of MMR will facilitate future studies on the immunomodulatory effects of alkylating agents in relevant DNA repair contexts, which will be vital for optimizing combinations with ICB. © 2025 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Validation of mismatch repair knockouts and <i>in vitro</i> sensitivity to alkylating agents</p><p><b>Basic Protocol 2</b>: Stereotaxic injection of isogenic SB28 cells in female C57BL/6J mice and <i>in vivo</i> treatment</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 2","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-02-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143481527","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}