{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,8]],"date-time":"2026-09-08T23:40:31Z","timestamp":1788910831233,"version":"build-2803163510"},"reference-count":29,"publisher":"Wiley","issue":"9","license":[{"start":{"date-parts":[[2024,9,12]],"date-time":"2024-09-12T00:00:00Z","timestamp":1726099200000},"content-version":"vor","delay-in-days":11,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Current Protocols"],"published-print":{"date-parts":[[2024,9]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>Cultured mammalian spermatogonial stem cells (SSCs), also known as germline stem cells (GSCs), hold great promise for applications such as fertility preservation, gene therapy, and animal breeding, particularly in conjunction with accurate gene editing. Although the <jats:italic>in vitro<\/jats:italic> development of mouse GSC (mGSC) lines, and gene\u2010targeting procedures for such lines, were initially established about two decades ago, it remains challenging for beginners to efficiently accomplish these tasks, partly because mGSCs proliferate more slowly and are more resistant to lipid\u2010mediated gene transfection than pluripotent stem cells (PSCs). Meanwhile, methods for mGSC culture and gene editing have been evolving constantly to become simpler and more efficient. Here, we describe how to develop mGSC lines from small mouse testis samples and how to carry out gene knock\u2010in in these cells using CRISPR\/Cas9 technology, detailing three basic protocols that constitute a streamlined procedure. Using these simple and efficient procedures, site\u2010specific knock\u2010in mGSC lines can be obtained in 3 months. We hope that these protocols will help researchers use genetically modified GSCs to explore scientific questions of interest and to accumulate experience for application to GSC research in other mammalian species. \u00a9 2024 Wiley Periodicals LLC.<\/jats:p><jats:p><jats:bold>Basic Protocol 1<\/jats:bold>: Establishment of mouse GSCs lines from small testicular samples<\/jats:p><jats:p><jats:bold>Basic Protocol 2<\/jats:bold>: Preparation of plasmids for gene knock\u2010in using the CRISPR\/Cas9 system<\/jats:p><jats:p><jats:bold>Basic Protocol 3<\/jats:bold>: Establishment of gene knock\u2010in mGSC lines by electroporation gene delivery<\/jats:p>","DOI":"10.1002\/cpz1.70002","type":"journal-article","created":{"date-parts":[[2024,9,12]],"date-time":"2024-09-12T14:43:23Z","timestamp":1726152203000},"source":"Crossref","is-referenced-by-count":3,"title":["A Simple and Efficient Procedure for Developing Mouse Germline Stem Cell Lines with Gene Knock\u2010in via CRISPR\/Cas9 Technology"],"prefix":"10.1002","volume":"4","author":[{"given":"Yang","family":"Wang","sequence":"first","affiliation":[{"name":"State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology Chinese Academy of Sciences  Beijing China"},{"name":"Institute for Stem Cell and Regeneration Chinese Academy of Sciences  Beijing China"},{"name":"Beijing Institute for Stem Cell and Regenerative Medicine  Beijing China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shuaitao","family":"Hu","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology Chinese Academy of Sciences  Beijing China"},{"name":"Institute for Stem Cell and Regeneration Chinese Academy of Sciences  Beijing China"},{"name":"Beijing Institute for Stem Cell and Regenerative Medicine  Beijing China"},{"name":"Savaid Medical School University of Chinese Academy of Sciences  Beijing China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1795-2852","authenticated-orcid":false,"given":"Chunsheng","family":"Han","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology Chinese Academy of Sciences  Beijing China"},{"name":"Institute for Stem Cell and Regeneration Chinese Academy of Sciences  Beijing China"},{"name":"Beijing Institute for Stem Cell and Regenerative Medicine  Beijing China"},{"name":"Savaid Medical School University of Chinese Academy of Sciences  Beijing 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