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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

Author
item ASADA, M - Nagasaki University
item YAHATA, K - Nagasaki University
item HAKIMI, H - Nagasaki University
item YOKOYAMA, N - Obihiro University Of Agriculture And Veterinary Medicine
item IGARASHI, I - Obihiro University Of Agriculture And Veterinary Medicine
item KANEKO, O - Nagasaki University
item Suarez, Carlos
item KAWAZU, S - Obihiro University Of Agriculture And Veterinary Medicine

Submitted to: PLoS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/27/2015
Publication Date: 5/11/2015
Citation: Asada, M., Yahata, K., Hakimi, H., Yokoyama, N., Igarashi, I., Kaneko, O., Suarez, C.E., Kawazu, S. 2015. Transfection of babesia bovis by double selection with WR99210 and blasticidin-S and its application for functional analysis of thioredoxin peroxidase-1. PLoS One. doi: 10.1371/journal.pone.0125993.

Interpretive Summary: 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 double transfection system 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.

Technical 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 double 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 double 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.