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- Title
Transfection of Babesia bovis by Double Selection with WR99210 and Blasticidin-S and Its Application for Functional Analysis of Thioredoxin Peroxidase-1.
- Authors
Asada, Masahito; Yahata, Kazuhide; Hakimi, Hassan; Yokoyama, Naoaki; Igarashi, Ikuo; Kaneko, Osamu; Suarez, Carlos E.; Kawazu, Shin-ichiro
- Abstract
Genetic manipulation is an essential technique to analyze gene function; however, limited methods are available for Babesia bovis, a causative pathogen of the globally important cattle disease, bovine babesiosis. To date, two stable transfection systems have been developed for B. bovis, using selectable markers blasticidin-S deaminase (bsd) or human dihydrofolate reductase (hdhfr). In this work, we combine these two selectable markers in a sequential transfection system. Specifically, a parent transgenic B. bovis line which episomally expresses green fluorescent protein (GFP) and human dihydrofolate reductase (hDHFR), was transfected with a plasmid encoding a fusion protein consisting of red fluorescent protein (RFP) and blasticidin-S deaminase (BSD). Selection with WR99210 and blasticidin-S resulted in the emergence of parasites double positive for GFP and RFP. We then applied this method to complement gene function in a parasite line in which thioredoxin peroxidase-1 (Bbtpx-1) gene was knocked out using hDHFR as a selectable marker. A plasmid was constructed harboring both RFP-BSD and Bbtpx-1 expression cassettes, and transfected into a Bbtpx-1 knockout (KO) parasite. Transfectants were independently obtained by two transfection methods, episomal transfection and genome integration. Complementation of Bbtpx-1 resulted in full recovery of resistance to nitrosative stress, via the nitric oxide donor sodium nitroprusside, which was impaired in the Bbtpx-1 KO parasites. In conclusion, we developed a sequential transfection method in B. bovis and subsequently applied this technique in a gene complementation study. This method will enable broader genetic manipulation of Babesia toward enhancing our understanding of the biology of this parasite.
- Subjects
GENE transfection; BABESIA; THIOREDOXIN; PEROXIDASE; TETRAHYDROFOLATE dehydrogenase
- Publication
PLoS ONE, 2015, Vol 10, Issue 5, p1
- ISSN
1932-6203
- Publication type
Article
- DOI
10.1371/journal.pone.0125993