{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,17]],"date-time":"2026-06-17T17:58:07Z","timestamp":1781719087326,"version":"3.54.5"},"reference-count":43,"publisher":"Public Library of Science (PLoS)","issue":"6","license":[{"start":{"date-parts":[[2026,6,17]],"date-time":"2026-06-17T00:00:00Z","timestamp":1781654400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100009141","name":"Cancer League of Colorado","doi-asserted-by":"publisher","award":["220552-SF"],"award-info":[{"award-number":["220552-SF"]}],"id":[{"id":"10.13039\/100009141","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100006383","name":"Children\u2019s Hospital Colorado Research Institute","doi-asserted-by":"publisher","award":["R9010L"],"award-info":[{"award-number":["R9010L"]}],"id":[{"id":"10.13039\/100006383","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS One"],"abstract":"<jats:p>\n                    The leading cause of brain cancer\u2013related death in children is diffuse midline glioma (DMG). A particularly aggressive DMG subtype is pontine DMG (formerly diffuse intrinsic pontine glioma, DIPG), which is caused by the histone mutation H3.3K27M. Because of its diffuse growth and location in a critical brainstem structure, therapeutic options are limited, and pontine DMG is considered universally fatal. The lack of appropriate animal models has hindered our understanding of the developmental origins and progression of pontine DMG, which in turn has limited the development of effective therapeutics. To address this barrier, several labs have developed mouse\n                    <jats:italic>in utero<\/jats:italic>\n                    electroporation approaches to express canonical DMG mutant oncogenes in the developing pons. In this manuscript and accompanying protocol, we describe a modified\n                    <jats:italic>in utero<\/jats:italic>\n                    electroporation strategy to generate DMG tumors in the developing mouse pons. Our protocol incorporates a single plasmid construct that expresses canonical DMG oncogenes, eliminating the need to co-electroporate multiple plasmids. We also employ a triple-electrode configuration to precisely target neural progenitors lining the fourth ventricle, which give rise to cells in the pons. As the embryos continue to develop\n                    <jats:italic>in utero<\/jats:italic>\n                    and postnatally, they form large, diffuse brainstem tumors with molecular characteristics of pediatric pontine DMG, allowing us to model the formation and progression of this deadly pediatric brain cancer.\n                  <\/jats:p>","DOI":"10.1371\/journal.pone.0351079","type":"journal-article","created":{"date-parts":[[2026,6,17]],"date-time":"2026-06-17T17:27:31Z","timestamp":1781717251000},"page":"e0351079","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":0,"title":["Modeling diffuse midline glioma through triple-electrode in utero electroporation of the developing mouse pons"],"prefix":"10.1371","volume":"21","author":[{"given":"Madisen S.","family":"Mason","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hosbaldo","family":"Morales Murillo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Madisyn G.","family":"Dudek","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Samantha E.","family":"Maher","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4478-5634","authenticated-orcid":true,"given":"Santos J.","family":"Franco","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2026,6,17]]},"reference":[{"key":"pone.0351079.ref001","doi-asserted-by":"crossref","DOI":"10.1093\/neuonc\/nou223","article-title":"Alex\u2019s lemonade stand foundation infant and childhood primary brain and central nervous system tumors diagnosed in the United States in 2007\u20132011","volume":"16","author":"QT Ostrom","year":"2014","journal-title":"Neuro-Oncology"},{"issue":"6","key":"pone.0351079.ref002","doi-asserted-by":"crossref","first-page":"863","DOI":"10.1097\/PAS.0000000000001897","article-title":"Diffuse midline gliomas with histone H3 K27M mutation in adults and children: A retrospective series of 164 cases","volume":"46","author":"L Zheng","year":"2022","journal-title":"Am J Surg Pathol"},{"key":"pone.0351079.ref003","doi-asserted-by":"crossref","first-page":"858148","DOI":"10.3389\/fonc.2022.858148","article-title":"Prognostic implication of patient age in H3K27M-mutant midline gliomas","volume":"12","author":"HG Vuong","year":"2022","journal-title":"Front Oncol"},{"issue":"3","key":"pone.0351079.ref004","doi-asserted-by":"crossref","first-page":"241","DOI":"10.1016\/S1470-2045(06)70615-5","article-title":"Diffuse brainstem glioma in children: Critical review of clinical trials","volume":"7","author":"D Hargrave","year":"2006","journal-title":"Lancet Oncol"},{"key":"pone.0351079.ref005","doi-asserted-by":"crossref","first-page":"1082062","DOI":"10.3389\/fonc.2022.1082062","article-title":"Pediatric diffuse midline glioma H3K27- altered: A complex clinical and biological landscape behind a neatly defined tumor type","volume":"12","author":"SG Vallero","year":"2023","journal-title":"Front Oncol"},{"key":"pone.0351079.ref006","doi-asserted-by":"crossref","first-page":"2064","DOI":"10.3390\/diagnostics12092064","article-title":"Pediatric diffuse midline gliomas: An unfinished puzzle","volume":"12","author":"V Di Ruscio","year":"2022","journal-title":"Diagnostics"},{"issue":"8","key":"pone.0351079.ref007","doi-asserted-by":"crossref","first-page":"1231","DOI":"10.1093\/neuonc\/noab106","article-title":"The 2021 WHO Classification of Tumors of the Central Nervous System: A summary","volume":"23","author":"DN Louis","year":"2021","journal-title":"Neuro Oncol"},{"issue":"20","key":"pone.0351079.ref008","doi-asserted-by":"crossref","first-page":"3451","DOI":"10.3390\/cancers16203451","article-title":"H3K27me3 loss in central nervous system tumors: Diagnostic, prognostic, and therapeutic implications","volume":"16","author":"G Angelico","year":"2024","journal-title":"Cancers (Basel)"},{"issue":"1","key":"pone.0351079.ref009","doi-asserted-by":"crossref","first-page":"1262","DOI":"10.1038\/s41467-019-09140-x","article-title":"H3K27M induces defective chromatin spread of PRC2-mediated repressive H3K27me2\/me3 and is essential for glioma tumorigenesis","volume":"10","author":"AS Harutyunyan","year":"2019","journal-title":"Nat Commun"},{"issue":"4","key":"pone.0351079.ref010","doi-asserted-by":"crossref","DOI":"10.1016\/j.ccell.2017.08.017","article-title":"Integrated molecular meta-analysis of 1,000 pediatric high-grade and diffuse intrinsic pontine Glioma","volume":"32","author":"A Mackay","year":"2017","journal-title":"Cancer Cell"},{"issue":"6386","key":"pone.0351079.ref011","doi-asserted-by":"crossref","first-page":"331","DOI":"10.1126\/science.aao4750","article-title":"Developmental and oncogenic programs in H3K27M gliomas dissected by single-cell RNA-seq","volume":"360","author":"MG Filbin","year":"2018","journal-title":"Science"},{"issue":"20","key":"pone.0351079.ref012","doi-asserted-by":"crossref","first-page":"6219","DOI":"10.1158\/0008-5472.CAN-13-1491","article-title":"Novel oncogenic PDGFRA mutations in pediatric high-grade gliomas","volume":"73","author":"BS Paugh","year":"2013","journal-title":"Cancer Res"},{"issue":"6134","key":"pone.0351079.ref013","doi-asserted-by":"crossref","first-page":"857","DOI":"10.1126\/science.1232245","article-title":"Inhibition of PRC2 activity by a gain-of-function H3 mutation found in pediatric glioblastoma","volume":"340","author":"PW Lewis","year":"2013","journal-title":"Science"},{"issue":"9","key":"pone.0351079.ref014","doi-asserted-by":"crossref","first-page":"1681","DOI":"10.1002\/glia.24189","article-title":"A novel mouse model of diffuse midline glioma initiated in neonatal oligodendrocyte progenitor cells highlights cell-of-origin dependent effects of H3K27M","volume":"70","author":"Y Tomita","year":"2022","journal-title":"Glia"},{"issue":"1","key":"pone.0351079.ref015","doi-asserted-by":"crossref","DOI":"10.1016\/j.ccell.2018.11.015","article-title":"Histone H3.3 K27M accelerates spontaneous brainstem glioma and drives restricted changes in bivalent gene expression","volume":"35","author":"JD Larson","year":"2019","journal-title":"Cancer Cell"},{"issue":"3","key":"pone.0351079.ref016","doi-asserted-by":"crossref","DOI":"10.1016\/j.ccell.2020.02.002","article-title":"Mutant ACVR1 arrests glial cell differentiation to drive tumorigenesis in pediatric gliomas","volume":"37","author":"J Fortin","year":"2020","journal-title":"Cancer Cell"},{"key":"pone.0351079.ref017","doi-asserted-by":"crossref","DOI":"10.1093\/neuonc\/noad073.060","article-title":"DIPG-13. New immunocompetent genetically-engineered mouse models of pediatric-type diffuse high-grade gliomas reveal mechanisms of tumor initiation and progression","volume":"25","author":"R Tian","year":"2023","journal-title":"Neuro-Oncology"},{"issue":"1","key":"pone.0351079.ref018","doi-asserted-by":"crossref","first-page":"1023","DOI":"10.1038\/s41467-019-08823-9","article-title":"ACVR1 R206H cooperates with H3.1K27M in promoting diffuse intrinsic pontine glioma pathogenesis","volume":"10","author":"CM Hoeman","year":"2019","journal-title":"Nat Commun"},{"issue":"6","key":"pone.0351079.ref019","doi-asserted-by":"crossref","first-page":"2548","DOI":"10.1158\/0008-5472.CAN-09-2503","article-title":"Preclinical evaluation of radiation and perifosine in a genetically and histologically accurate model of brainstem glioma","volume":"70","author":"OJ Becher","year":"2010","journal-title":"Cancer Res"},{"issue":"5","key":"pone.0351079.ref020","doi-asserted-by":"crossref","DOI":"10.1016\/j.ccell.2017.09.014","article-title":"H3.3K27M Cooperates with Trp53 Loss and PDGFRA Gain in Mouse Embryonic Neural Progenitor Cells to Induce Invasive High-Grade Gliomas","volume":"32","author":"M Pathania","year":"2017","journal-title":"Cancer Cell"},{"issue":"3","key":"pone.0351079.ref021","first-page":"381","article-title":"Generation of diffuse intrinsic pontine glioma mouse models by brainstem-targeted in utero electroporation","volume":"22","author":"SK Patel","year":"2020","journal-title":"Neuro Oncol"},{"issue":"3","key":"pone.0351079.ref022","doi-asserted-by":"crossref","DOI":"10.1016\/j.ccell.2025.12.020","article-title":"IGSF11-VISTA is a critical and targetable immune checkpoint axis in diffuse midline glioma","volume":"44","author":"R Collot","year":"2026","journal-title":"Cancer Cell"},{"issue":"7","key":"pone.0351079.ref023","doi-asserted-by":"crossref","first-page":"1592","DOI":"10.1158\/2159-8290.CD-23-0004","article-title":"A Compendium of Syngeneic, Transplantable Pediatric High-Grade Glioma Models Reveals Subtype-Specific Therapeutic Vulnerabilities","volume":"13","author":"M McNicholas","year":"2023","journal-title":"Cancer Discov"},{"issue":"1","key":"pone.0351079.ref024","article-title":"Generation of immunocompetent syngeneic allograft mouse models for pediatric diffuse midline glioma","volume":"4","author":"A du Chatinier","year":"2022","journal-title":"Neurooncol Adv"},{"key":"pone.0351079.ref025","doi-asserted-by":"crossref","DOI":"10.1093\/neuonc\/noad073.082","article-title":"DIPG-35. Identifying driver-specific vulnerabilities in paediatric high grade glioma subtypes","volume":"25","author":"A De Cola","year":"2023","journal-title":"Neuro-Oncology"},{"issue":"10","key":"pone.0351079.ref026","doi-asserted-by":"crossref","first-page":"5313","DOI":"10.1172\/JCI133310","article-title":"Everolimus improves the efficacy of dasatinib in PDGFR\u03b1-driven glioma","volume":"130","author":"Z Miklja","year":"2020","journal-title":"J Clin Invest"},{"issue":"1","key":"pone.0351079.ref027","doi-asserted-by":"crossref","first-page":"54","DOI":"10.1093\/neuonc\/noac141","article-title":"Therapeutic targeting of prenatal pontine ID1 signaling in diffuse midline glioma","volume":"25","author":"D Messinger","year":"2023","journal-title":"Neuro Oncol"},{"issue":"6","key":"pone.0351079.ref028","article-title":"Histone H3.3G34-Mutant Interneuron Progenitors Co-opt PDGFRA for Gliomagenesis","volume":"183","author":"CCL Chen","year":"2020","journal-title":"Cell"},{"issue":"23","key":"pone.0351079.ref029","doi-asserted-by":"crossref","first-page":"5237","DOI":"10.1523\/JNEUROSCI.3392-17.2018","article-title":"The dorsal wave of neocortical oligodendrogenesis begins embryonically and requires multiple sources of sonic hedgehog","volume":"38","author":"CC Winkler","year":"2018","journal-title":"J Neurosci"},{"issue":"5","key":"pone.0351079.ref030","doi-asserted-by":"crossref","DOI":"10.1242\/dev.170068","article-title":"Lmx1a drives Cux2 expression in the cortical hem through activation of a conserved intronic enhancer","volume":"146","author":"SP Fregoso","year":"2019","journal-title":"Development"},{"key":"pone.0351079.ref031","doi-asserted-by":"crossref","DOI":"10.1523\/ENEURO.0434-18.2019","article-title":"Csmd2 is a synaptic transmembrane protein that interacts with PSD-95 and is required for neuronal maturation","volume":"6","author":"MA Gutierrez","year":"2019","journal-title":"eNeuro"},{"key":"pone.0351079.ref032","article-title":"Notch signaling plays a dual role in regulating the neuron-to-oligodendrocyte switch in the developing dorsal forebrain","author":"LN Tran","year":"2023","journal-title":"J Neurosci"},{"key":"pone.0351079.ref033","doi-asserted-by":"crossref","first-page":"21","DOI":"10.1007\/978-1-4939-3014-2_2","article-title":"In utero electroporation methods in the study of cerebral cortical development","author":"I Mart\u00ednez-Garay","year":"2016","journal-title":"Prenatal and postnatal determinants of development"},{"key":"pone.0351079.ref034","first-page":"100027","article-title":"In utero cortical electroporation of plasmids in the mouse embryo. STAR Protocols","volume":"1","author":"G Meyer-Dilhet","year":"2020"},{"key":"pone.0351079.ref035","doi-asserted-by":"crossref","DOI":"10.17504\/protocols.io.5jyl8dy7dg2w\/v1","article-title":"In uteroelectroporation of mouse pons via the 4th ventricle v1","author":"MS Mason","year":"2025"},{"key":"pone.0351079.ref036","doi-asserted-by":"crossref","first-page":"82","DOI":"10.3389\/fnmol.2020.00082","article-title":"Double UP: A dual color, internally controlled platform for in utero knockdown or overexpression","volume":"13","author":"RJ Taylor","year":"2020","journal-title":"Front Mol Neurosci"},{"issue":"3","key":"pone.0351079.ref037","doi-asserted-by":"crossref","first-page":"399","DOI":"10.1038\/nprot.2016.014","article-title":"Targeted in vivo genetic manipulation of the mouse or rat brain by in utero electroporation with a triple-electrode probe","volume":"11","author":"J Szczurkowska","year":"2016","journal-title":"Nat Protoc"},{"key":"pone.0351079.ref038","doi-asserted-by":"crossref","first-page":"11628","DOI":"10.1038\/ncomms11628","article-title":"Identification of proliferative progenitors associated with prominent postnatal growth of the pons","volume":"7","author":"RA Lindquist","year":"2016","journal-title":"Nat Commun"},{"issue":"11","key":"pone.0351079.ref039","doi-asserted-by":"crossref","first-page":"2786","DOI":"10.1111\/febs.16426","article-title":"Regulation of early cerebellar development","volume":"290","author":"ED Lowenstein","year":"2023","journal-title":"FEBS J"},{"key":"pone.0351079.ref040","doi-asserted-by":"crossref","first-page":"789","DOI":"10.1007\/s12311-015-0724-2","article-title":"Consensus paper: Cerebellar development","volume":"15","author":"K Leto","year":"2016","journal-title":"Cerebellum"},{"key":"pone.0351079.ref041","doi-asserted-by":"crossref","first-page":"10","DOI":"10.3389\/fnana.2019.00010","article-title":"Time for radical changes in brain stem nomenclature-applying the lessons from developmental gene patterns","volume":"13","author":"C Watson","year":"2019","journal-title":"Front Neuroanat"},{"issue":"3","key":"pone.0351079.ref042","doi-asserted-by":"crossref","first-page":"1045","DOI":"10.1523\/JNEUROSCI.4499-05.2006","article-title":"Molecular and morphological heterogeneity of neural precursors in the mouse neocortical proliferative zones","volume":"26","author":"JS Gal","year":"2006","journal-title":"J Neurosci"},{"key":"pone.0351079.ref043","article-title":"A pipeline using bilateral in utero electroporation to interrogate genetic influences on rodent behavior","author":"AL Comer","year":"2020","journal-title":"J Vis Exp"}],"container-title":["PLOS One"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0351079","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,6,17]],"date-time":"2026-06-17T17:27:37Z","timestamp":1781717257000},"score":1,"resource":{"primary":{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0351079"}},"subtitle":[],"editor":[{"given":"Xiaojun","family":"Ren","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"editor"}]}],"short-title":[],"issued":{"date-parts":[[2026,6,17]]},"references-count":43,"journal-issue":{"issue":"6","published-online":{"date-parts":[[2026,6,17]]}},"URL":"https:\/\/doi.org\/10.1371\/journal.pone.0351079","relation":{},"ISSN":["1932-6203"],"issn-type":[{"value":"1932-6203","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,6,17]]}}}