{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,26]],"date-time":"2025-10-26T14:29:54Z","timestamp":1761488994664},"reference-count":35,"publisher":"American Society for Microbiology","issue":"11","license":[{"start":{"date-parts":[[2012,11,1]],"date-time":"2012-11-01T00:00:00Z","timestamp":1351728000000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["Eukaryot Cell"],"published-print":{"date-parts":[[2012,11]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n          <jats:p>\n            Eukaryotic microbes are highly diverse, and many lineages remain poorly studied. One such lineage, the diplomonads, a group of binucleate heterotrophic flagellates, has been studied mainly due to the impact of\n            <jats:named-content content-type=\"genus-species\">Giardia intestinalis<\/jats:named-content>\n            , an intestinal, diarrhea-causing parasite in humans and animals. Here we describe the development of a stable transfection system for use in\n            <jats:named-content content-type=\"genus-species\">Spironucleus salmonicida<\/jats:named-content>\n            , a diplomonad that causes systemic spironucleosis in salmonid fish. We designed vectors in cassette format carrying epitope tags for localization (3\u00d7HA [where HA is hemagglutinin], 2\u00d7\n            <jats:named-content content-type=\"genus-species\">Escherichia coli<\/jats:named-content>\n            OmpF linker and mouse langerin fusion sequence [2\u00d7OLLAS], 3\u00d7MYC) and purification of proteins (2\u00d7 Strep-Tag II\u2013FLAG tandem-affinity purification tag or streptavidin binding peptide\u2013glutathione\n            <jats:italic>S<\/jats:italic>\n            -transferase [SBP-GST]) under the control of native or constitutive promoters. Three selectable gene markers, puromycin acetyltransferase (\n            <jats:italic>pac<\/jats:italic>\n            ), blasticidin\n            <jats:italic>S<\/jats:italic>\n            -deaminase (\n            <jats:italic>bsr<\/jats:italic>\n            ), and neomycin phosphotransferase (\n            <jats:italic>nptII<\/jats:italic>\n            ), were successfully applied for the generation of stable transfectants. Site-specific integration on the\n            <jats:named-content content-type=\"genus-species\">S. salmonicida<\/jats:named-content>\n            chromosome was shown to be possible using the\n            <jats:italic>bsr<\/jats:italic>\n            resistance gene. We epitope tagged six proteins and confirmed their expression by Western blotting. Next, we demonstrated the utility of these vectors by recording the subcellular localizations of the six proteins by laser scanning confocal microscopy. Finally, we described the creation of an\n            <jats:named-content content-type=\"genus-species\">S. salmonicida<\/jats:named-content>\n            double transfectant suitable for colocalization studies. The transfection system described herein and the imminent completion of the\n            <jats:named-content content-type=\"genus-species\">S. salmonicida<\/jats:named-content>\n            genome will make it possible to use comparative genomics as an investigative tool to explore specific, as well as general, diplomonad traits, benefiting research on both\n            <jats:named-content content-type=\"genus-species\">Giardia<\/jats:named-content>\n            and\n            <jats:named-content content-type=\"genus-species\">Spironucleu<\/jats:named-content>\n            s.\n          <\/jats:p>","DOI":"10.1128\/ec.00179-12","type":"journal-article","created":{"date-parts":[[2012,9,15]],"date-time":"2012-09-15T11:29:59Z","timestamp":1347708599000},"page":"1353-1361","update-policy":"http:\/\/dx.doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":14,"title":["Stable Transfection of the Diplomonad Parasite Spironucleus salmonicida"],"prefix":"10.1128","volume":"11","author":[{"given":"Jon","family":"Jerlstr\u00f6m-Hultqvist","sequence":"first","affiliation":[{"name":"Department of Cell and Molecular Biology, BMC, Uppsala University, Uppsala, Sweden"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Elin","family":"Einarsson","sequence":"additional","affiliation":[{"name":"Department of Cell and Molecular Biology, BMC, Uppsala University, Uppsala, Sweden"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Staffan G.","family":"Sv\u00e4rd","sequence":"additional","affiliation":[{"name":"Department of Cell and Molecular Biology, BMC, Uppsala University, Uppsala, Sweden"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"235","reference":[{"key":"e_1_3_3_2_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0960-9822(03)00003-4"},{"key":"e_1_3_3_3_2","doi-asserted-by":"publisher","DOI":"10.1186\/1471-2164-8-51"},{"key":"e_1_3_3_4_2","doi-asserted-by":"publisher","DOI":"10.1111\/j.1432-1033.1994.tb18710.x"},{"key":"e_1_3_3_5_2","doi-asserted-by":"publisher","DOI":"10.1007\/978-0-387-74021-8_2"},{"key":"e_1_3_3_6_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.femsle.2004.01.043"},{"key":"e_1_3_3_7_2","doi-asserted-by":"publisher","DOI":"10.1186\/1471-2180-8-71"},{"key":"e_1_3_3_8_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1000560"},{"key":"e_1_3_3_9_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2010.01.032"},{"key":"e_1_3_3_10_2","doi-asserted-by":"publisher","DOI":"10.1002\/pmic.200700038"},{"key":"e_1_3_3_11_2","doi-asserted-by":"publisher","DOI":"10.1128\/EC.00190-10"},{"key":"e_1_3_3_12_2","doi-asserted-by":"publisher","DOI":"10.1016\/0092-8674(87)90576-9"},{"key":"e_1_3_3_13_2","doi-asserted-by":"publisher","DOI":"10.1091\/mbc.e07-11-1156"},{"key":"e_1_3_3_14_2","doi-asserted-by":"publisher","DOI":"10.1083\/jcb.200608041"},{"key":"e_1_3_3_15_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1002167"},{"key":"e_1_3_3_16_2","doi-asserted-by":"publisher","DOI":"10.1186\/1471-2164-11-543"},{"key":"e_1_3_3_17_2","doi-asserted-by":"publisher","DOI":"10.1128\/EC.00092-12"},{"key":"e_1_3_3_18_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.ijpara.2008.04.012"},{"key":"e_1_3_3_19_2","doi-asserted-by":"publisher","DOI":"10.14411\/fp.2007.036"},{"key":"e_1_3_3_20_2","doi-asserted-by":"publisher","DOI":"10.1111\/j.1550-7408.2006.00144.x"},{"key":"e_1_3_3_21_2","doi-asserted-by":"publisher","DOI":"10.1038\/nmeth.2047"},{"key":"e_1_3_3_22_2","doi-asserted-by":"publisher","DOI":"10.1093\/oxfordjournals.molbev.a025832"},{"key":"e_1_3_3_23_2","doi-asserted-by":"publisher","DOI":"10.1016\/0166-6851(96)02636-9"},{"key":"e_1_3_3_24_2","doi-asserted-by":"publisher","DOI":"10.1111\/j.1365-2761.2010.01213.x"},{"key":"e_1_3_3_25_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.1143837"},{"key":"e_1_3_3_26_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.jim.2007.10.012"},{"key":"e_1_3_3_27_2","doi-asserted-by":"publisher","DOI":"10.3354\/dao045197"},{"key":"e_1_3_3_28_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.cub.2005.01.037"},{"key":"e_1_3_3_29_2","doi-asserted-by":"publisher","DOI":"10.1002\/j.1460-2075.1984.tb02297.x"},{"key":"e_1_3_3_30_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.cub.2008.12.032"},{"key":"e_1_3_3_31_2","doi-asserted-by":"publisher","DOI":"10.1186\/1471-2164-11-258"},{"key":"e_1_3_3_32_2","doi-asserted-by":"publisher","DOI":"10.3354\/dao039135"},{"key":"e_1_3_3_33_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0166-6851(97)00225-9"},{"key":"e_1_3_3_34_2","doi-asserted-by":"publisher","DOI":"10.1007\/s00253-002-1158-6"},{"key":"e_1_3_3_35_2","doi-asserted-by":"publisher","DOI":"10.1128\/EC.05262-11"},{"key":"e_1_3_3_36_2","doi-asserted-by":"publisher","DOI":"10.1242\/jcs.93.3.491"}],"container-title":["Eukaryotic 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