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Many members of the C1q\/TNF superfamily function as synaptic organizers, shaping synapse assembly and maintenance. Among them, C1QL3 plays a putative role in trans\u2010synaptic adhesion and modulation of synaptic strength, but the lack of a reliable antibody to detect it has severely limited the ability to map its endogenous localization and study its biochemical properties. Here, we present a novel epitope\u2010tagged knock\u2010in mouse line (\n                    <jats:italic>C1ql3<\/jats:italic>\n                    <jats:sup>2HA<\/jats:sup>\n                    ), in which two hemagglutinin (HA) epitopes were inserted near the N\u2010terminus of the endogenous C1QL3 protein. This model enables purification, detection, and subcellular localization of native C1QL3 protein (C1QL3\u20102HA) with high specificity, eliminating the need for overexpression or custom antibodies. We validated that\n                    <jats:italic>C1ql3<\/jats:italic>\n                    <jats:sup>2HA<\/jats:sup>\n                    mice maintain normal mRNA expression, biochemical properties, and behavior. Using native PAGE, we determined the endogenous oligomeric state of C1QL3\u20102HA. Brain\u2010wide light\u2010sheet microscopy uncovered an expanded neuroanatomical map of C1QL3\u20102HA expression, including newly identified populations in cortical and subcortical regions as well as the retina. Dual immunohistochemistry confirmed cell\u2010type\u2010specific expression patterns, and super\u2010resolution STED microscopy localized C1QL3\u20102HA to hippocampal mossy fiber synapses, positioned between pre\u2010 and postsynaptic markers, supporting its hypothesized role in trans\u2010synaptic complexes. This knock\u2010in mouse line is a valuable tool for studying the anatomical, molecular, and synaptic biology of C1QL3 in all cellular\/tissue contexts, enabling future studies into its potential roles in the nervous system and beyond.\n                  <\/jats:p>","DOI":"10.1002\/cne.70193","type":"journal-article","created":{"date-parts":[[2026,8,7]],"date-time":"2026-08-07T12:20:29Z","timestamp":1786105229000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Brain\u2010Wide Mapping and Synaptic Localization of C1QL3 Using a Novel Epitope\u2010Tagged Knock\u2010In Mouse"],"prefix":"10.1002","volume":"534","author":[{"suffix":"IV","given":"William P.","family":"Armstrong","sequence":"first","affiliation":[{"name":"Department of Physiology and Neurobiology University of Connecticut  Storrs Connecticut USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"James M.","family":"Salvatore","sequence":"additional","affiliation":[{"name":"Department of Physiology and Neurobiology University of Connecticut  Storrs Connecticut USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Matthew J.","family":"Sticco","sequence":"additional","affiliation":[{"name":"Department of Neuroscience University of Connecticut Health  Farmington Connecticut USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Keaven","family":"Caro","sequence":"additional","affiliation":[{"name":"Department of Neuroscience University of Connecticut Health  Farmington Connecticut USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"J. Wesley","family":"Maddox","sequence":"additional","affiliation":[{"name":"Department of Neuroscience University of Texas at Austin  Austin Texas USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Angie","family":"Huang","sequence":"additional","affiliation":[{"name":"Department of Neuroscience University of Texas at Austin  Austin Texas USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Brenna C.","family":"McAllister","sequence":"additional","affiliation":[{"name":"Department of Neuroscience University of Connecticut Health  Farmington Connecticut USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Christopher B.","family":"O'Connell","sequence":"additional","affiliation":[{"name":"Advanced Light Microscopy Facility, Center for Open Research Resources and Equipment University of Connecticut  Storrs Connecticut 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