Chimeric fluorescent reporter as a tool for generation of transgenic Eimeria (Apicomplexa, Coccidia) strains with stage specific reporter gene expression

Progress in transfection of Eimeria sporozoites leads to transformed oocysts, however the output of mutants after passages in the host animals is low. Further enrichment of transgenic oocysts was dependent on fluorescent activated cell sorting and could not be achieved by drug selection. In this study, we fused the Toxoplasma gondii DHFR-TSm2m3 pyrimethamine resistance gene with the yellow fluorescent protein (YFP) encoding sequence to provide continuous pyrimethamine resistance and fluorescence in the Eimeria parasite from a single transcript. The permanent YFP signal of transgenic parasites allows differentiating transgenic parasites from wild type parasites throughout the entire life cycle. The output of transformed oocysts increased up to more than 30% after initial transfection and completion of the life cycle in the host animal. Within three passages under pyrimethamine treatment, a strain with 100% transformed sporulated oocysts of the parasite could be isolated. This new method provides the potential to produce and monitor transgenic Eimeria strains without additional fluorescence activated cell sorting (FACS). The chimeric fluorescent reporter can be utilized as a continuous internal control for plasmids containing stage specific promoter. By this means we utilized an Eimeria tenella gamogony gene specific regulatory sequence to confer macrogamont specific tandem dimer tomato (tdtomato) reporter gene expression in Eimeria nieschulzi.

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► Generation of 100% transformed sporulated Eimeria nieschulzi oocysts.
► Selection and control of resistance via yfp tagged pyrimethamine resistance gene.
► Gamogony specific promoter of Etgam56 (E. tenella) is functional in E. nieschulzi.
► Gamogony specific expression of fluorescent tandem dimer tomato (tdtomato).
► Constant visible transgenicity and accessory stage specific tdtomato expression.