{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,11]],"date-time":"2026-10-11T03:10:08Z","timestamp":1791688208980,"version":"4.3.4"},"reference-count":36,"publisher":"Springer Science and Business Media LLC","issue":"1","license":[{"start":{"date-parts":[[2024,6,5]],"date-time":"2024-06-05T00:00:00Z","timestamp":1717545600000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"},{"start":{"date-parts":[[2024,6,5]],"date-time":"2024-06-05T00:00:00Z","timestamp":1717545600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"}],"content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["BMC Genomics"],"abstract":"<jats:title>Abstract<\/jats:title><jats:sec>\n                <jats:title>Background<\/jats:title>\n                <jats:p>Transgenic (Tg) mice are widely used in biomedical research, and they are typically generated by injecting transgenic DNA cassettes into pronuclei of one-cell stage zygotes. Such animals often show unreliable expression of the transgenic DNA, one of the major reasons for which is random insertion of the transgenes. We previously developed a method called \u201cpronuclear injection-based targeted transgenesis\u201d (PITT), in which DNA constructs are directed to insert at pre-designated genomic loci. PITT was achieved by pre-installing so called landing pad sequences (such as heterotypic <jats:italic>LoxP<\/jats:italic> sites or <jats:italic>attP<\/jats:italic> sites) to create seed mice and then injecting <jats:italic>Cre<\/jats:italic> recombinase or <jats:italic>PhiC31<\/jats:italic> integrase mRNAs along with a compatible donor plasmid into zygotes derived from the seed mice. PITT and its subsequent version, improved PITT (<jats:italic>i<\/jats:italic>-PITT), overcome disadvantages of conventional Tg mice such as lack of consistent and reliable expression of the cassettes among different Tg mouse lines, and the PITT approach is superior in terms of cost and labor. One of the limitations of PITT, particularly using <jats:italic>Cre<\/jats:italic>-mRNA, is that the approach cannot be used for insertion of conditional expression cassettes using Cre-<jats:italic>LoxP<\/jats:italic> site-specific recombination. This is because the <jats:italic>LoxP<\/jats:italic> sites in the donor plasmids intended for achieving conditional expression of the transgene will interfere with the PITT recombination reaction with <jats:italic>LoxP<\/jats:italic> sites in the landing pad.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Results<\/jats:title>\n                <jats:p>To enable the <jats:italic>i<\/jats:italic>-PITT method to insert a conditional expression cassette, we modified the approach by simultaneously using <jats:italic>PhiC31o<\/jats:italic> and <jats:italic>FLPo<\/jats:italic> mRNAs. We demonstrate the strategy by creating a model containing a conditional expression cassette at the <jats:italic>Rosa26<\/jats:italic> locus with an efficiency of 13.7%. We also demonstrate that inclusion of <jats:italic>FLPo<\/jats:italic> mRNA excludes the insertion of vector backbones in the founder mice.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Conclusions<\/jats:title>\n                <jats:p>Simultaneous use of <jats:italic>PhiC31<\/jats:italic> and <jats:italic>FLP<\/jats:italic> in <jats:italic>i<\/jats:italic>-PITT approach allows insertion of donor plasmids containing Cre-<jats:italic>loxP<\/jats:italic>-based conditional expression cassettes.<\/jats:p>\n              <\/jats:sec>","DOI":"10.1186\/s12864-024-10250-0","type":"journal-article","created":{"date-parts":[[2024,6,5]],"date-time":"2024-06-05T15:02:23Z","timestamp":1717599743000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Targeted insertion of conditional expression cassettes into the mouse genome using the modified i-PITT"],"prefix":"10.1186","volume":"25","author":[{"given":"Hiromi","family":"Miura","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ayaka","family":"Nakamura","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aki","family":"Kurosaki","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ai","family":"Kotani","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Masaru","family":"Motojima","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Keiko","family":"Tanaka","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shigeru","family":"Kakuta","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sanae","family":"Ogiwara","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuhsuke","family":"Ohmi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hirotaka","family":"Komaba","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Samantha L.P.","family":"Schilit","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Cynthia C.","family":"Morton","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Channabasavaiah B.","family":"Gurumurthy","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Masato","family":"Ohtsuka","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2024,6,5]]},"reference":[{"key":"10250_CR1","doi-asserted-by":"publisher","first-page":"7380","DOI":"10.1073\/pnas.77.12.7380","volume":"77","author":"JW Gordon","year":"1980","unstructured":"Gordon JW, Scangos GA, Plotkin DJ, Barbosa JA, Ruddle FH. 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This study was carried out in compliance with the ARRIVE guidelines.","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":"HK has received grant support from Kyowa Kirin and honoraria from Chugai Pharmaceutical, Kissei Pharmaceutical, Kyowa Kirin, Ono Pharmaceutical, and Sanwa Kagaku Kenkyusho.","order":4,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"568"}}