Development of a stable transgenic Theileria equi parasite expressing an enhanced green fluorescent protein/blasticidin S deaminase

Abstract Theileria equi, an intraerythrocytic protozoan parasite, causes equine piroplasmosis, a disease which negatively impacts the global horse industry. Genetic manipulation is one of the research tools under development as a control method for protozoan parasites, but this technique needs to be...

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Autores principales:	Bumduuren Tuvshintulga, Arifin Budiman Nugraha, Tomoka Mizutani, Mingming Liu, Takahiro Ishizaki, Thillaiampalam Sivakumar, Xuenan Xuan, Naoaki Yokoyama, Ikuo Igarashi
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/0486893e623c49ac89feded910611d5a
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spelling	oai:doaj.org-article:0486893e623c49ac89feded910611d5a2021-12-02T17:15:33ZDevelopment of a stable transgenic Theileria equi parasite expressing an enhanced green fluorescent protein/blasticidin S deaminase10.1038/s41598-021-88594-w2045-2322https://doaj.org/article/0486893e623c49ac89feded910611d5a2021-04-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-88594-whttps://doaj.org/toc/2045-2322Abstract Theileria equi, an intraerythrocytic protozoan parasite, causes equine piroplasmosis, a disease which negatively impacts the global horse industry. Genetic manipulation is one of the research tools under development as a control method for protozoan parasites, but this technique needs to be established for T. equi. Herein, we report on the first development of a stable transgenic T. equi line expressing enhanced green fluorescent protein/blasticidin S deaminase (eGFP/BSD). To express the exogenous fusion gene in T. equi, regulatory regions of the elongation factor-1 alpha (ef-1α) gene were identified in T. equi. An eGFP/BSD-expression cassette containing the ef-1α gene promoter and terminator regions was constructed and integrated into the T. equi genome. On day 9 post-transfection, blasticidin-resistant T. equi emerged. In the clonal line of T. equi obtained by limiting dilution, integration of the eGFP/BSD-expression cassette was confirmed in the designated B-locus of the ef-1α gene via PCR and Southern blot analyses. Parasitaemia dynamics between the transgenic and parental T. equi lines were comparable in vitro. The eGFP/BSD-expressing transgenic T. equi and the methodology used to generate it offer new opportunities for better understanding of T. equi biology, with the add-on possibility of discovering effective control methods against equine piroplasmosis.Bumduuren TuvshintulgaArifin Budiman NugrahaTomoka MizutaniMingming LiuTakahiro IshizakiThillaiampalam SivakumarXuenan XuanNaoaki YokoyamaIkuo IgarashiNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-10 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Bumduuren Tuvshintulga Arifin Budiman Nugraha Tomoka Mizutani Mingming Liu Takahiro Ishizaki Thillaiampalam Sivakumar Xuenan Xuan Naoaki Yokoyama Ikuo Igarashi Development of a stable transgenic Theileria equi parasite expressing an enhanced green fluorescent protein/blasticidin S deaminase
description	Abstract Theileria equi, an intraerythrocytic protozoan parasite, causes equine piroplasmosis, a disease which negatively impacts the global horse industry. Genetic manipulation is one of the research tools under development as a control method for protozoan parasites, but this technique needs to be established for T. equi. Herein, we report on the first development of a stable transgenic T. equi line expressing enhanced green fluorescent protein/blasticidin S deaminase (eGFP/BSD). To express the exogenous fusion gene in T. equi, regulatory regions of the elongation factor-1 alpha (ef-1α) gene were identified in T. equi. An eGFP/BSD-expression cassette containing the ef-1α gene promoter and terminator regions was constructed and integrated into the T. equi genome. On day 9 post-transfection, blasticidin-resistant T. equi emerged. In the clonal line of T. equi obtained by limiting dilution, integration of the eGFP/BSD-expression cassette was confirmed in the designated B-locus of the ef-1α gene via PCR and Southern blot analyses. Parasitaemia dynamics between the transgenic and parental T. equi lines were comparable in vitro. The eGFP/BSD-expressing transgenic T. equi and the methodology used to generate it offer new opportunities for better understanding of T. equi biology, with the add-on possibility of discovering effective control methods against equine piroplasmosis.
format	article
author	Bumduuren Tuvshintulga Arifin Budiman Nugraha Tomoka Mizutani Mingming Liu Takahiro Ishizaki Thillaiampalam Sivakumar Xuenan Xuan Naoaki Yokoyama Ikuo Igarashi
author_facet	Bumduuren Tuvshintulga Arifin Budiman Nugraha Tomoka Mizutani Mingming Liu Takahiro Ishizaki Thillaiampalam Sivakumar Xuenan Xuan Naoaki Yokoyama Ikuo Igarashi
author_sort	Bumduuren Tuvshintulga
title	Development of a stable transgenic Theileria equi parasite expressing an enhanced green fluorescent protein/blasticidin S deaminase
title_short	Development of a stable transgenic Theileria equi parasite expressing an enhanced green fluorescent protein/blasticidin S deaminase
title_full	Development of a stable transgenic Theileria equi parasite expressing an enhanced green fluorescent protein/blasticidin S deaminase
title_fullStr	Development of a stable transgenic Theileria equi parasite expressing an enhanced green fluorescent protein/blasticidin S deaminase
title_full_unstemmed	Development of a stable transgenic Theileria equi parasite expressing an enhanced green fluorescent protein/blasticidin S deaminase
title_sort	development of a stable transgenic theileria equi parasite expressing an enhanced green fluorescent protein/blasticidin s deaminase
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/0486893e623c49ac89feded910611d5a
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Development of a stable transgenic Theileria equi parasite expressing an enhanced green fluorescent protein/blasticidin S deaminase

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