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It has also recently been reported to be an important member of the host intestinal microbiota. Despite significant advances in our understanding of <jats:italic>Blastocystis<\/jats:italic> cell biology and host-parasite interactions, a genetic modification tool is absent. In this study, we successfully established a robust gene delivery protocol for <jats:italic>Blastocystis<\/jats:italic> subtype 7 (ST7) and ectopic protein expression was further tested using a high sensitivity nano-luciferase (Nluc) reporter system, with promoter regions from several genes. Among them, a strong promoter encompassing a region upstream of the legumain 5\u2032 UTR was identified. Using this promoter combined with the legumain 3\u2032 UTR, which contains a conserved, precise polyadenylation signal, a robust transient transfection technique was established for the first time in <jats:italic>Blastocystis<\/jats:italic>. This system was validated by ectopic expression of proteins harbouring specific localization signals. The establishment of a robust, reproducible gene modification system for <jats:italic>Blastocystis<\/jats:italic> is a significant advance for <jats:italic>Blastocystis<\/jats:italic> research both <jats:italic>in vitro<\/jats:italic> and <jats:italic>in vivo<\/jats:italic>. This technique will spearhead further research to understand the parasite\u2019s biology, its role in health and disease, along with novel ways to combat the parasite.<\/jats:p>","DOI":"10.1038\/s41598-019-39094-5","type":"journal-article","created":{"date-parts":[[2019,2,28]],"date-time":"2019-02-28T11:05:29Z","timestamp":1551351929000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":10,"title":["Successful Genetic Transfection of the Colonic Protistan Parasite Blastocystis for Reliable Expression of Ectopic Genes"],"prefix":"10.1038","volume":"9","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-1662-999X","authenticated-orcid":false,"given":"Feng-Jun","family":"Li","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5424-1905","authenticated-orcid":false,"given":"Anastasios D.","family":"Tsaousis","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tracy","family":"Purton","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Vincent T. 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