{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,11,5]],"date-time":"2026-11-05T12:32:58Z","timestamp":1793881978272,"version":"4.7.0"},"reference-count":15,"publisher":"Springer Science and Business Media LLC","issue":"1","license":[{"start":{"date-parts":[[2021,9,23]],"date-time":"2021-09-23T00:00:00Z","timestamp":1632355200000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"},{"start":{"date-parts":[[2021,9,23]],"date-time":"2021-09-23T00:00:00Z","timestamp":1632355200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"}],"funder":[{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["R01MH080047"],"award-info":[{"award-number":["R01MH080047"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["R35NS116804"],"award-info":[{"award-number":["R35NS116804"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["Mol Brain"],"published-print":{"date-parts":[[2021,12]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>\n                    The Cre\/LoxP-based conditional knockout technology is a powerful tool for gene function analysis that allows region- and time-specific gene manipulation. However, inserting a pair of LoxP cassettes to generate conditional knockout can be technically challenging and thus time- and resource-consuming. This study proposes an efficient, low-cost method to generate floxed mice using in vitro fertilization and the CRISPR-Cas9 system over two consecutive generations. This method allowed us to produce floxed mice targeting exons 5 and 6 of\n                    <jats:italic>CaMK1<\/jats:italic>\n                    in a short period of 125\u2009days, using only 16 mice. In addition, we directly edited the genome of fertilized eggs of mice with our target genetic background, C57BL\/6\u2009N, to eliminate additional backcrossing steps. We confirmed that the genome of the generated floxed mice was responsive to the Cre protein. This low-cost, time-saving method for generating conditional knockout will facilitate comprehensive, tissue-specific genome analyses.\n                  <\/jats:p>","DOI":"10.1186\/s13041-021-00859-7","type":"journal-article","created":{"date-parts":[[2021,9,23]],"date-time":"2021-09-23T19:05:23Z","timestamp":1632423923000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":15,"title":["Rapid generation of conditional knockout mice using the CRISPR-Cas9 system and electroporation for neuroscience research"],"prefix":"10.1186","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-9394-7089","authenticated-orcid":false,"given":"Hirofumi","family":"Nishizono","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuki","family":"Hayano","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yoshihisa","family":"Nakahata","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yasuhito","family":"Ishigaki","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ryohei","family":"Yasuda","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2021,9,23]]},"reference":[{"key":"859_CR1","doi-asserted-by":"publisher","first-page":"1317","DOI":"10.1016\/S0092-8674(00)81826-7","volume":"87","author":"JZ Tsien","year":"1996","unstructured":"Tsien JZ, Chen DF, Gerber D, Tom C, Mercer EH, Anderson DJ, et al. Subregion- and cell type-restricted gene knockout in mouse brain. Cell. 1996;87:1317\u201326. https:\/\/doi.org\/10.1016\/S0092-8674(00)81826-7.","journal-title":"Cell"},{"key":"859_CR2","doi-asserted-by":"publisher","first-page":"507","DOI":"10.1038\/nrg1619","volume":"6","author":"MR Capecchi","year":"2005","unstructured":"Capecchi MR. Gene targeting in mice: Functional analysis of the mammalian genome for the twenty-first century. Nat Rev Genet. 2005;6:507\u201312. https:\/\/doi.org\/10.1038\/nrg1619.","journal-title":"Nat Rev Genet"},{"key":"859_CR3","doi-asserted-by":"publisher","first-page":"22","DOI":"10.4252\/wjsc.v1.i1.22","volume":"1","author":"AC Carstea","year":"2009","unstructured":"Carstea AC, Pirity MK, Dinnyes A. Germline competence of mouse ES and iPS cell lines: Chimera technologies and genetic background. World J Stem Cells. 2009;1:22\u20139. https:\/\/doi.org\/10.4252\/wjsc.v1.i1.22.","journal-title":"World J Stem Cells"},{"key":"859_CR4","doi-asserted-by":"publisher","first-page":"e0142755","DOI":"10.1371\/journal.pone.0142755","volume":"10","author":"T Kaneko","year":"2015","unstructured":"Kaneko T, Mashimo T. Simple genome editing of rodent intact embryos by electroporation. PLOS ONE. 2015;10:e0142755. https:\/\/doi.org\/10.1371\/journal.pone.0142755.","journal-title":"PLOS ONE"},{"key":"859_CR5","doi-asserted-by":"publisher","first-page":"423","DOI":"10.1534\/genetics.115.176594","volume":"200","author":"W Qin","year":"2015","unstructured":"Qin W, Dion SL, Kutny PM, Zhang Y, Cheng AW, Jillette NL, et al. Efficient CRISPR\/Cas9-mediated genome editing in mice by zygote electroporation of nuclease. Genetics. 2015;200:423\u201330. https:\/\/doi.org\/10.1534\/genetics.115.176594.","journal-title":"Genetics"},{"key":"859_CR6","doi-asserted-by":"publisher","first-page":"2452","DOI":"10.1038\/s41596-019-0187-x","volume":"14","author":"CB Gurumurthy","year":"2019","unstructured":"Gurumurthy CB, Sato M, Nakamura A, Inui M, Kawano N, Islam MA, et al. Creation of CRISPR-based germline-genome-engineered mice without ex vivo handling of zygotes by i-GONAD. Nat Protoc. 2019;14:2452\u201382. https:\/\/doi.org\/10.1038\/s41596-019-0187-x.","journal-title":"Nat Protoc"},{"key":"859_CR7","doi-asserted-by":"publisher","first-page":"18","DOI":"10.3389\/fgeed.2020.602970","volume":"2","author":"H Nishizono","year":"2020","unstructured":"Nishizono H, Yasuda R, Laviv T. Methodologies and challenges for CRISPR\/Cas9 mediated genome editing of the mammalian brain. Front Genome Ed. 2020;2:18. https:\/\/doi.org\/10.3389\/fgeed.2020.602970.","journal-title":"Front Genome Ed"},{"key":"859_CR8","doi-asserted-by":"publisher","first-page":"92","DOI":"10.1186\/s13059-017-1220-4","volume":"18","author":"RM Quadros","year":"2017","unstructured":"Quadros RM, Miura H, Harms DW, Akatsuka H, Sato T, Aida T, et al. Easi-CRISPR: A robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins. Genome Biol. 2017;18:92. https:\/\/doi.org\/10.1186\/s13059-017-1220-4.","journal-title":"Genome Biol"},{"key":"859_CR9","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1038\/s41598-017-08496-8","volume":"7","author":"T Horii","year":"2017","unstructured":"Horii T, Morita S, Kimura M, Terawaki N, Shibutani M, Hatada I. Efficient generation of conditional knockout mice via sequential introduction of lox sites. 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Genome Res. 2021;31:121\u201330. https:\/\/doi.org\/10.1101\/gr.265439.120.","journal-title":"Genome Res"},{"key":"859_CR14","doi-asserted-by":"publisher","first-page":"1341","DOI":"10.1016\/j.theriogenology.2016.04.076","volume":"86","author":"T Takeo","year":"2016","unstructured":"Takeo T, Nakagata N. Immunotherapy using inhibin antiserum enhanced the efficacy of equine chorionic gonadotropin on superovulation in major inbred and outbred mice strains. Theriogenology. 2016;86:1341\u20136. https:\/\/doi.org\/10.1016\/j.theriogenology.2016.04.076.","journal-title":"Theriogenology"},{"key":"859_CR15","doi-asserted-by":"publisher","first-page":"9076","DOI":"10.1093\/nar\/gkv901","volume":"43","author":"C Zhang","year":"2015","unstructured":"Zhang C, Myers CA, Qi Z, Mitra RD, Corbo JC, Havranek JJ. Redesign of the monomer-monomer interface of Cre recombinase yields an obligate heterotetrameric complex. Nucleic Acids Res. 2015;43:9076\u201385. https:\/\/doi.org\/10.1093\/nar\/gkv901.","journal-title":"Nucleic Acids Res"}],"container-title":["Molecular Brain"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/link.springer.com\/content\/pdf\/10.1186\/s13041-021-00859-7.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/link.springer.com\/article\/10.1186\/s13041-021-00859-7\/fulltext.html","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/link.springer.com\/content\/pdf\/10.1186\/s13041-021-00859-7.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2021,9,23]],"date-time":"2021-09-23T19:12:21Z","timestamp":1632424341000},"score":1,"resource":{"primary":{"URL":"https:\/\/molecularbrain.biomedcentral.com\/articles\/10.1186\/s13041-021-00859-7"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,9,23]]},"references-count":15,"journal-issue":{"issue":"1","published-print":{"date-parts":[[2021,12]]}},"alternative-id":["859"],"URL":"https:\/\/doi.org\/10.1186\/s13041-021-00859-7","relation":{"has-preprint":[{"id-type":"doi","id":"10.1101\/2021.05.09.443330","asserted-by":"object"}]},"ISSN":["1756-6606"],"issn-type":[{"value":"1756-6606","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,9,23]]},"assertion":[{"value":"9 May 2021","order":1,"name":"received","label":"Received","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"13 September 2021","order":2,"name":"accepted","label":"Accepted","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"23 September 2021","order":3,"name":"first_online","label":"First Online","group":{"name":"ArticleHistory","label":"Article History"}},{"order":1,"name":"Ethics","group":{"name":"EthicsHeading","label":"Declarations"}},{"value":"All animal experiments\nwere conducted in accordance with the guidelines and rules of the Institutional\nAnimal Care and Use Committee of Max Planck Florida Institute for Neuroscience.\nThe experimental protocol was approved by the same committee (Approval Number:\n19-001).","order":2,"name":"Ethics","group":{"name":"EthicsHeading","label":"Ethics approval and consent to participate"}},{"value":"Not applicable.","order":3,"name":"Ethics","group":{"name":"EthicsHeading","label":"Consent for publication"}},{"value":"The authors declare that they have no competing interests.","order":4,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"148"}}