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Stable transfection technique has been reported in many protozoan parasites over the past few years. However, in vivo transient and stable transfection method has not been established for <jats:italic>Babesia microti<\/jats:italic>. Here, for the first time, we present a method of transient as well as stable transfection of the <jats:italic>Babesia microti (B. microti)<\/jats:italic> in the in vivo conditions. We have identified a novel promoter of <jats:italic>B. microti.<\/jats:italic> We also demonstrated that <jats:italic>Plasmodium berghei<\/jats:italic> DHFR promoter is recognized and functional in <jats:italic>B. microti.<\/jats:italic> We show that BM-CTQ41297 promoter control the expression of two genes, which are present on either side and thus represents a bi-functional promoter in <jats:italic>B. microti.<\/jats:italic> The predicted promoter activity values using Promoter 2.0 program is higher for BM- CTQ41297 promoter than strong promoters such as \u03b2-actin, ef-1\u03b2, and many other promoters. Furthermore, we discovered a non-essential locus for the genetic manipulation of the parasite, allowing us to stably integrate foreign genes; <jats:italic>GFP<\/jats:italic>, <jats:italic>mCherry<\/jats:italic>, into the <jats:italic>B. microti.<\/jats:italic> The transfection using an electroporation method and genetic manipulation of <jats:italic>B. microti<\/jats:italic> is now achievable and it is possible to obtain transfected viable parasites under in vivo growing conditions. The growth curve analysis of transfected and WT <jats:italic>B. microti<\/jats:italic> are similar indicating no defects in the transgenic parasites. This study will enable other researchers in understanding the <jats:italic>B. microti<\/jats:italic> biology, host modulation and diverse parasite developmental stages using reverse genetics and holds great potential to identify novel drug targets and vaccine development.<\/jats:p>","DOI":"10.1038\/s41598-020-72489-3","type":"journal-article","created":{"date-parts":[[2020,9,24]],"date-time":"2020-09-24T10:04:50Z","timestamp":1600941890000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":14,"title":["Establishment of a stable transfection method in Babesia microti and identification of a novel bidirectional promoter of Babesia microti"],"prefix":"10.1038","volume":"10","author":[{"given":"Dabbu Kumar","family":"Jaijyan","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kavitha","family":"Govindasamy","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jyoti","family":"Singh","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Shreya","family":"Bhattacharya","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Agam Prasad","family":"Singh","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"297","published-online":{"date-parts":[[2020,9,24]]},"reference":[{"key":"72489_CR1","doi-asserted-by":"publisher","first-page":"1057","DOI":"10.1007\/s15010-013-0526-8","volume":"41","author":"A Hildebrandt","year":"2013","unstructured":"Hildebrandt, A. et al. 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