{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,29]],"date-time":"2026-09-29T13:30:57Z","timestamp":1790688657036,"version":"4.1.0"},"reference-count":67,"publisher":"MDPI AG","issue":"2","license":[{"start":{"date-parts":[[2025,1,22]],"date-time":"2025-01-22T00:00:00Z","timestamp":1737504000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000005","name":"Department of Defense U.S. Army Medical Research and Materiel Command","doi-asserted-by":"publisher","award":["W81XWH-18-1-0319"],"award-info":[{"award-number":["W81XWH-18-1-0319"]}],"id":[{"id":"10.13039\/100000005","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Vaccines"],"abstract":"<jats:p>Background\/Objectives: Incidence of vector-borne diseases, including rickettsioses and anaplasmosis, has been increasing in many parts of the world. The obligate intracellular nature of rickettsial pathogens has hindered the development of robust genetic tools for the study of gene function and the identification of therapeutic targets. Transposon mutagenesis has contributed to recent progress in the identification of virulence factors in this important group of pathogens. Methods: Combining the efficiency of the himar1 transposon method with a recombinase-mediated system, we aimed to develop a genetic tool enabling the exchange of the transposon with a cassette encoding non-native sequences. Results: This approach was used in Rickettsia parkeri to insert a himar1 transposon encoding fluorescent protein and antibiotic resistance genes for visualization and selection, flanked by mismatched loxP sites to enable subsequent recombinase-mediated cassette exchange (RMCE). RMCE mediated by a plasmid-encoded Cre recombinase was then employed to replace the transposon with a different cassette containing alternate fluorescent and selection markers and epitopes of Anaplasma phagocytophilum antigens. The resulting genetically modified R. parkeri was trialed as a live-attenuated vaccine against spotted fever rickettsiosis and anaplasmosis in mice. Conclusions: The use of this system provides a well-established and relatively efficient way of inserting non-native sequences into the rickettsial genome, with applications for the study of gene function and vaccine development.<\/jats:p>","DOI":"10.3390\/vaccines13020109","type":"journal-article","created":{"date-parts":[[2025,1,22]],"date-time":"2025-01-22T05:50:53Z","timestamp":1737525053000},"page":"109","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Development of a Recombinase-Mediated Cassette Exchange System for Gene Knockout and Expression of Non-Native Gene Sequences in Rickettsia"],"prefix":"10.3390","volume":"13","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-6814-5669","authenticated-orcid":false,"given":"Benjamin","family":"Cull","sequence":"first","affiliation":[{"name":"Department of Entomology, College of Food, Agricultural and Natural Resource Sciences, University of Minnesota, St. Paul, MN 55108, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nicole Y.","family":"Burkhardt","sequence":"additional","affiliation":[{"name":"Department of Entomology, College of Food, Agricultural and Natural Resource Sciences, University of Minnesota, St. Paul, MN 55108, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Benedict S.","family":"Khoo","sequence":"additional","affiliation":[{"name":"Division of Environmental Health Sciences, School of Public Health, University of Minnesota, Minneapolis, MN 55455, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8738-112X","authenticated-orcid":false,"given":"Jonathan D.","family":"Oliver","sequence":"additional","affiliation":[{"name":"Division of Environmental Health Sciences, School of Public Health, University of Minnesota, Minneapolis, MN 55455, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xin-Ru","family":"Wang","sequence":"additional","affiliation":[{"name":"Department of Entomology, College of Food, Agricultural and Natural Resource Sciences, University of Minnesota, St. Paul, MN 55108, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0002-0833-4032","authenticated-orcid":false,"given":"Lisa D.","family":"Price","sequence":"additional","affiliation":[{"name":"Department of Entomology, College of Food, Agricultural and Natural Resource Sciences, University of Minnesota, St. Paul, MN 55108, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3881-737X","authenticated-orcid":false,"given":"Kamil","family":"Khanipov","sequence":"additional","affiliation":[{"name":"Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, TX 77555, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rong","family":"Fang","sequence":"additional","affiliation":[{"name":"Department of Pathology, University of Texas Medical Branch, Galveston, TX 77555, USA"},{"name":"Center for Biodefense and Emerging Infectious Diseases, University of Texas Medical Branch, Galveston, TX 77555, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ulrike G.","family":"Munderloh","sequence":"additional","affiliation":[{"name":"Department of Entomology, College of Food, Agricultural and Natural Resource Sciences, University of Minnesota, St. Paul, MN 55108, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,1,22]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"213","DOI":"10.1016\/j.idc.2018.10.010","article-title":"The Rickettsioses: A Practical Update","volume":"33","author":"Blanton","year":"2019","journal-title":"Infect. 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