{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,4]],"date-time":"2026-08-04T14:55:13Z","timestamp":1785855313575,"version":"3.56.0"},"reference-count":35,"publisher":"Wiley","issue":"7","license":[{"start":{"date-parts":[[2026,7,21]],"date-time":"2026-07-21T00:00:00Z","timestamp":1784592000000},"content-version":"vor","delay-in-days":20,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,7,1]],"date-time":"2026-07-01T00:00:00Z","timestamp":1782864000000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Current Protocols"],"published-print":{"date-parts":[[2026,7]]},"abstract":"Abstract<\/jats:title>\n \n Nanobodies are small but specific heavy chain\u2013only antibody fragments. Their small size, relative stability, and ability to access difficult to reach deep\u2010tissue antigens makes them valuable research, diagnostic, and therapeutic tools. Nanobodies are derived from the variable heavy (VH) domain of heavy chain\u2013only antibodies that are unique to camelids, including alpacas, llamas, and camels. The approaches employed to produce nanobodies, have been evolving and expanding since the initial discovery of heavy chain\u2013only antibodies 30 years ago. Traditional nanobody development involves camelid immunization with a soluble, purified protein, followed by blood collection and processing, enrichment for potent nanobody sequences, and eventual expression and purification of candidate nanobodies for testing and validation. Alternative nanobody generation strategies aim to identify novel nanobodies utilizing synthetic or animal\u2010derived na\u00efve nanobody libraries in combination with phage\u2010, yeast surface\u2010, or ribosome displays for nanobody selection. This article outlines a novel protocol series for nanobody production using a commercially available transgenic \u201cnanomouse\u201d, engineered to produce heavy chain\u2013only antibodies containing camelid VH domains from alpacas, dromedaries, and Bactrian camels. These protocols will cover the following aspects: (1) Nanomouse breeding, genotyping, and colony establishment; (2) Nanomouse immunization and tissue collection; (3) RNA extraction from nanomouse immune cells isolated from blood and tissues; (4) Generation and amplification of VHH DNA from nanomouse cDNA; (5) Digestion of VHH DNA and ligation into a phagemid expression vector; (6) Preparation of a screenable\n Escherichia coli<\/jats:italic>\n TG1\u2010based phagemid library; (7) Antigen\u2010driven VHH selection using phage display; (8) Single colony VHH ELISA screening; (9) Sequencing of ELISA hits and candidate VHH sequence identification; (10) Geneblock design of candidate VHH and Gibson Assembly into a nanobody expression vector; and (11) Nanobody over\u2010expression and purification. We provide a comprehensive toolkit to facilitate nanobody development and make it more accessible to the greater research community. \u00a9 2026 The Author(s).\n Current Protocols<\/jats:italic>\n published by Wiley Periodicals LLC.\n <\/jats:p>\n \n Basic Protocol 1<\/jats:bold>\n : Nanomouse breeding, genotyping, and colony establishment\n <\/jats:p>\n \n Basic Protocol 2<\/jats:bold>\n : Nanomouse immunization and tissue collection\n <\/jats:p>\n \n Basic Protocol 3<\/jats:bold>\n : RNA extraction from nanomouse immune cells isolated from blood and tissues\n <\/jats:p>\n \n Basic Protocol 4<\/jats:bold>\n : Generation and amplification of VHH DNA from nanomouse cDNA\n <\/jats:p>\n \n Basic Protocol 5<\/jats:bold>\n : Digestion of VHH DNA and ligation into a phagemid expression vector\n <\/jats:p>\n \n Basic Protocol 6<\/jats:bold>\n : Preparation of a screenable\n E. coli<\/jats:italic>\n TG1\u2010based phagemid library\n <\/jats:p>\n \n Basic Protocol 7<\/jats:bold>\n : Antigen\u2010driven VHH selection using phage display\n <\/jats:p>\n \n Basic Protocol 8<\/jats:bold>\n : Single colony VHH enzyme\u2010linked immunosorbent assay (ELISA) screening\n <\/jats:p>\n \n Basic Protocol 9<\/jats:bold>\n : Sequencing of ELISA hits and candidate VHH sequence identification\n <\/jats:p>\n \n Basic Protocol 10<\/jats:bold>\n : Geneblock design of candidate VHH and Gibson Assembly into a nanobody expression vector\n <\/jats:p>\n \n Basic Protocol 11<\/jats:bold>\n : Nanobody over\u2010expression and purification\n <\/jats:p>","DOI":"10.1002\/cpz1.70432","type":"journal-article","created":{"date-parts":[[2026,7,21]],"date-time":"2026-07-21T17:51:56Z","timestamp":1784656316000},"source":"Crossref","is-referenced-by-count":0,"title":["High\u2010Throughput Isolation of Nanomouse\u2010Derived VHH Domains: A Practical Guide from Immunization to Nanobody Expression"],"prefix":"10.1002","volume":"6","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-0428-3469","authenticated-orcid":false,"given":"Tessa J.","family":"Casselman","sequence":"first","affiliation":[{"name":"Department of Molecular & Integrative Physiology University of Michigan Ann Arbor Michigan"},{"name":"These authors contributed equally to this work"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Asa W.","family":"Huffaker","sequence":"additional","affiliation":[{"name":"Department of Molecular & Integrative Physiology University of Michigan Ann Arbor Michigan"},{"name":"These authors contributed equally to this work"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9678-3462","authenticated-orcid":false,"given":"Kristie C.","family":"Mitchell","sequence":"additional","affiliation":[{"name":"Department of Molecular & Integrative Physiology University of Michigan Ann Arbor Michigan"},{"name":"These authors contributed equally to this work"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jamie E.","family":"Schnarrs","sequence":"additional","affiliation":[{"name":"Department of Molecular & Integrative Physiology University of Michigan Ann Arbor Michigan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sylvia","family":"Ni","sequence":"additional","affiliation":[{"name":"Department of Molecular & Integrative Physiology University of Michigan Ann Arbor Michigan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mary E.","family":"Skinner","sequence":"additional","affiliation":[{"name":"Department of Molecular & Integrative Physiology University of Michigan Ann Arbor Michigan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Matthias C.","family":"Truttmann","sequence":"additional","affiliation":[{"name":"Department of Molecular & Integrative Physiology University of Michigan Ann Arbor Michigan"},{"name":"Geriatrics Center 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