Abstract
piggyBac, a type II transposon that is useful for efficient transgenesis and insertional mutagenesis, has been used for effective and stable transfection in a wide variety of organisms. In this study we investigate the potential use of the piggyBac transposon system for forward genetics studies in the apicomplexan parasite Eimeria tenella. Using the restriction enzyme-mediated integration (REMI) method, E. tenella sporozoites were electroporated with a donor plasmid containing the enhanced yellow fluorescent protein (EYFP) gene flanked by piggyBac inverted terminal repeats (ITRs), an Asc I-linearized helper plasmid containing the transposase gene and the restriction enzyme Asc I. Subsequently, electroporated sporozoites were inoculated into chickens via the cloacal route and transfected progeny oocysts expressing EYFP were sorted by flow cytometry. A transgenic E. tenella population was selected by successive in vivo passage. Southern-blotting analysis showed that exogenous DNA containing the EYFP gene was integrated into the parasite genome at a limited number of integration sites and that the inserted part of the donor plasmid was the fragment located between the 5′ and 3′ ITRs as indicated by primer-specific PCR screening. Genome walking revealed that the insertion sites were TTAA-specific, which is consistent with the transposition characteristics of piggyBac.
Highlights
Avian coccidiosis caused by infection with one or more Eimeria species parasite incurs global economic losses of,£1,500 million annually [1]
Transfection with circular donor plasmid pHEA-Bac and the restriction enzyme Asc I resulted in fluorescent parasites in the in vitro cultures at a similar level to the cultures infected with co-transfected parasites (Fig. 2H) but did not result in any enhanced yellow fluorescent protein (EYFP) expression in parasites following in vivo infection of chickens
We report here that the piggyBac transposon system functions in E. tenella by transposing exogenous sequences into TTAA sites
Summary
Avian coccidiosis caused by infection with one or more Eimeria species parasite incurs global economic losses of ,£1,500 million annually [1]. A better understanding of the biology of Eimeria parasites is essential for the development of new strategies for effective control of avian coccidia. The development of protocols supporting genetic manipulation including transient and stable transfection of Eimeria tenella is beginning to provide complementary tools for functional genomic and transcriptomic analyses [6,7,8,9,10]. Despite these efforts functional genomic studies remain limited for Eimeria because of the lack of powerful and user-friendly molecular tools
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