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Here, we describe a shared method for the stable transfection of these two parasites using electroporation and blasticidin\/blasticidin deaminase as a selectable marker. Stably transfected<jats:italic>B<\/jats:italic>.<jats:italic>bigemina<\/jats:italic>and<jats:italic>B<\/jats:italic>.<jats:italic>bovis<\/jats:italic>were obtained using a common transfection plasmid targeting the enhanced<jats:italic>green fluorescent protein-BSD<\/jats:italic>(<jats:italic>egfp-bsd<\/jats:italic>) fusion gene into the<jats:italic>elongation factor-1\u03b1<\/jats:italic>(<jats:italic>ef-1\u03b1<\/jats:italic>) locus of<jats:italic>B<\/jats:italic>.<jats:italic>bigemina<\/jats:italic>and<jats:italic>B<\/jats:italic>.<jats:italic>bovis<\/jats:italic>under the control of the<jats:italic>B<\/jats:italic>.<jats:italic>bigemina ef-1\u03b1<\/jats:italic>promoter. Sequencing, Southern blotting, immunoblotting and immunofluorescence analysis of parasite-infected red blood cells, demonstrated that the<jats:italic>egfp-bsd<\/jats:italic>gene was expressed and stably integrated solely into the<jats:italic>ef-1\u03b1<\/jats:italic>locus of both,<jats:italic>B<\/jats:italic>.<jats:italic>bigemina<\/jats:italic>and<jats:italic>B<\/jats:italic>.<jats:italic>bovis<\/jats:italic>. Interestingly, heterologous<jats:italic>B<\/jats:italic>.<jats:italic>bigemina ef-1\u03b1<\/jats:italic>sequences were able to drive integration into the<jats:italic>B<\/jats:italic>.<jats:italic>bovis<\/jats:italic>genome by homologous recombination, and the stably integrated<jats:italic>B<\/jats:italic>.<jats:italic>bigemina ef-1\u03b1<\/jats:italic>-A promoter is fully functional in<jats:italic>B<\/jats:italic>.<jats:italic>bovis<\/jats:italic>. Collectively, the data provides a new tool for genetic analysis of these parasites, and suggests that the development of vaccine platform delivery systems based on transfected<jats:italic>B<\/jats:italic>.<jats:italic>bovis<\/jats:italic>and<jats:italic>B<\/jats:italic>.<jats:italic>bigemina<\/jats:italic>parasites using homologous and heterologous promoters is feasible.<\/jats:p>","DOI":"10.1038\/s41598-018-23010-4","type":"journal-article","created":{"date-parts":[[2018,4,11]],"date-time":"2018-04-11T10:27:12Z","timestamp":1523442432000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":19,"title":["Stable transformation of Babesia bigemina and Babesia bovis using a single transfection plasmid"],"prefix":"10.1038","volume":"8","author":[{"given":"Marta G.","family":"Silva","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Donald P.","family":"Knowles","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Monica L.","family":"Mazuz","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Brian M.","family":"Cooke","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Carlos E.","family":"Suarez","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"297","published-online":{"date-parts":[[2018,4,17]]},"reference":[{"key":"23010_CR1","doi-asserted-by":"publisher","first-page":"197","DOI":"10.1080\/00034983.1973.11686877","volume":"67","author":"DF Mahoney","year":"1973","unstructured":"Mahoney, D. F., Wright, I. G. & Mirre, G. B. Bovine babesiasis: the persistence of immunity to Babesia argentina and B. bigemina in calves (Bos taurus) after naturally acquired infection. Annals of tropical medicine and parasitology 67, 197\u2013203 (1973).","journal-title":"Annals of tropical medicine and parasitology"},{"key":"23010_CR2","doi-asserted-by":"publisher","first-page":"289","DOI":"10.1016\/j.ijpara.2008.08.006","volume":"39","author":"CE Suarez","year":"2009","unstructured":"Suarez, C. E. & McElwain, T. F. Stable expression of a GFP-BSD fusion protein in Babesia bovis merozoites. International journal for parasitology 39, 289\u2013297, https:\/\/doi.org\/10.1016\/j.ijpara.2008.08.006 (2009).","journal-title":"International journal for parasitology"},{"key":"23010_CR3","doi-asserted-by":"publisher","first-page":"205","DOI":"10.1016\/j.vetpar.2009.09.022","volume":"167","author":"CE Suarez","year":"2010","unstructured":"Suarez, C. E. & McElwain, T. F. 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