{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,2,27]],"date-time":"2026-02-27T05:48:18Z","timestamp":1772171298813,"version":"3.50.1"},"reference-count":39,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2022,4,6]],"date-time":"2022-04-06T00:00:00Z","timestamp":1649203200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["31930108, 31772729"],"award-info":[{"award-number":["31930108, 31772729"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Cell. Infect. Microbiol."],"abstract":"<jats:p>Genetic modification provides an invaluable molecular tool to dissect the biology and pathogenesis of pathogens. However, no report is available about the genetic modification of <jats:italic>Babesia duncani<\/jats:italic>, a pathogen responsible for human babesiosis that is widespread in North America, suggesting the necessity to develop a genetic manipulation method to improve the strategies for studying and understanding the biology of protozoan pathogens. The establishment of a genetic modification method requires promoters, selectable markers, and reporter genes. Here, the double-copy gene <jats:italic>elongation factor-1\u03b1<\/jats:italic> (<jats:italic>ef-1\u03b1<\/jats:italic>) and its promoters were amplified by conventional PCR and confirmed by sequencing. We established a transient transfection system by using the <jats:italic>ef-1\u03b1B<\/jats:italic> promoter and the reporter gene <jats:italic>mCherry<\/jats:italic> and achieved stable transfection through homologous recombination to integrate the selection marker <jats:italic>hDHFR-eGFP<\/jats:italic> into the parasite genome. The potential of this genetic modification method was tested by knocking out the <jats:italic>thioredoxin peroxidase-1<\/jats:italic> (<jats:italic>TPX-1<\/jats:italic>) gene, and under the drug pressure of 5\u00a0nM WR99210, 96.3% of the parasites were observed to express green fluorescence protein (eGFP) by flow cytometry at day 7 post-transfection. Additionally, the clone line of the <jats:italic>TPX-1<\/jats:italic> knockout parasite was successfully obtained by the limiting dilution method. This study provided a transfection method for <jats:italic>B. duncani<\/jats:italic>, which may facilitate gene function research and vaccine development of <jats:italic>B. duncani<\/jats:italic>.<\/jats:p>","DOI":"10.3389\/fcimb.2022.844498","type":"journal-article","created":{"date-parts":[[2022,4,6]],"date-time":"2022-04-06T06:45:48Z","timestamp":1649227548000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":12,"title":["Establishment of a Transient and Stable Transfection System for Babesia duncani Using a Homologous Recombination Strategy"],"prefix":"10.3389","volume":"12","author":[{"given":"Sen","family":"Wang","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Dongfang","family":"Li","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Fangwei","family":"Chen","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Weijun","family":"Jiang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Wanxin","family":"Luo","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Guan","family":"Zhu","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Junlong","family":"Zhao","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lan","family":"He","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1965","published-online":{"date-parts":[[2022,4,6]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"19974","DOI":"10.1074\/jbc.AC118.005771","article-title":"Establishment of a Continuous In Vitro Culture of Babesia Duncani in Human Erythrocytes Reveals Unusually High Tolerance to Recommended Therapies","volume":"293","author":"Abraham","year":"2018","journal-title":"J. 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