We have achieved stable expression of green fluorescent protein (GFP) in Babesia bovis by using the WR99210/human dihydrofolate reductase (DHFR) gene selection system. A GFP-expression plasmid with a dhfr expression cassette (DHFR-gfp) was constructed and transfected into B. bovis by nucleofection. Following WR99210 selection, a GFP-fluorescent parasite population was obtained and the fluorescent parasite was maintained for more than 7 months under WR99210 drug pressure. The DHFR-gfp was used to construct a small circular chromosome and to target gene disruption in the parasite. For construction of the small circular chromosome (DHFR-gfp-Bbcent2), the putative centromere region of B. bovis chromosome 2 (Bbcent2) was cloned and inserted into the DHFR-gfp plasmid. Addition of Bbcent2 to the DHFR-gfp plasmid improved its segregation efficiency during parasite multiplication and GFP-expressing parasites were maintained for more than 2 months without drug pressure. For targeted disruption of a B. bovis gene we attempted to knockout the thioredoxin peroxidase-1 (TPx-1) gene (a single-copy 2-Cys peroxiredoxin gene, Tbtpx-1) by homologous recombination. To generate the targeting construct (DHFR-gfp-Bbtpx1KO), 5' and 3' portions of Bbtpx-1 were cloned into the DHFR-gfp plasmid. Following nucleofection, WR99210 selection and cloning, a GFP-fluorescent parasite population was obtained. Integration of the construct into the Bbtpx-1 locus was confirmed by PCR. The absence of Bbtpx-1 mRNA and protein were verified by reverse transcription PCR and western blot analysis/indirect immunofluorescence assay, respectively. This is the first report of targeted gene disruption of a Babesia gene. These advances in the methodology of genetic manipulation in B. bovis will facilitate functional analysis of Babesia genomes and will improve our understanding of the basic biology of apicomplexan parasites.
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