Stable expression of green fluorescent protein and targeted disruption of thioredoxin peroxidase-1 gene in Babesia bovis with the WR99210/dhfr selection system

► Stable expression of GFP in Babesia bovis was achieved by using the WR99210/dhfr selection system. ► GFP-positive parasites having the plasmid with a dhfr expression cassette (DHFR-gfp) were maintained for more than 7 months under drug pressure. ► A small circular chromosome was constructed by using DHFR-gfp and a putative B. bovis centromere. ► The TPx-1 gene in B. bovis was successfully disrupted with DHFR-gfp by homologous recombination.