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Establishment of transient and stable transfection systems for Babesia ovata

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Title: Establishment of transient and stable transfection systems for Babesia ovata
Authors: Hakimi, Hassan Browse this author →KAKEN DB
Yamagishi, Junya Browse this author →KAKEN DB
Kegawa, Yuto Browse this author
Kaneko, Osamu Browse this author →KAKEN DB
Kawazu, Shin-ichiro Browse this author →KAKEN DB
Asada, Masahito Browse this author →KAKEN DB
Keywords: Babesia ovata
Bovine babesiosis
Double cross-over homologous recombination
Stable transfection
Issue Date: 23-May-2016
Publisher: BioMed Central
Journal Title: Parasites & vectors
Volume: 9
Issue: 1
Start Page: 171
Publisher DOI: 10.1186/s13071-016-1439-z
PMID: 27008652
Abstract: Background: Bovine babesiosis is a tick-borne disease caused by several species of Babesia which produce acute and fatal disease in cattle and affect livestock industry worldwide. Babesia ovata is a benign species widespread in east Asian countries and causes anemia, particularly in cattle which are co-infected with Theileria orientalis. The development of genetic manipulation methods is necessary to improve our understanding of the basic biology of protozoan pathogens toward a better control of disease. Such tools have not been developed for B. ovata, and are the aim of this study. Methods: In this study we transfected constructs that were designed to evaluate the ability of several B. ovata promoter candidates to drive expression of a reporter luciferase. We found that the elongation factor-1 alpha intergenic region (ef-1α IG) and the actin 5' non-coding region (NR) had highest promoter activities. To establish a stable transfection system, we generated a plasmid construct in which the ef-1α IG promoter drives gfp expression, and the actin 5' NR mediates expression of the selectable marker hdhfr. The plasmid was designed for episomal transfection, as well as to integrate by double cross-over homologous recombination into the ef-1α locus. Circular or linearized plasmid was transfected by electroporation into in vitro cultured B. ovata and retention of the plasmid was facilitated by drug selection with 5 nM WR99210 initiated 48 h after transfection. Results: After one-week cultivation with WR99210, GFP-expressing parasites were observed by fluorescence microscopy. Integration of the plasmid construct into the ef-1α locus was confirmed by PCR, Southern blot analysis, and sequencing of recombination sites. These results confirm successful development of a stable transfection system for B. ovata. Conclusion: The current study provides a fundamental molecular tool to aid in molecular and cellular studies of B. ovata.
Type: article
Appears in Collections:人獣共通感染症国際共同研究所 (International Institute for Zoonosis Control) > 雑誌発表論文等 (Peer-reviewed Journal Articles, etc)
国際連携研究教育局 : GI-CoRE (Global Institution for Collaborative Research and Education : GI-CoRE) > 雑誌発表論文等 (Peer-reviewed Journal Articles, etc)

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