Expression of the tandem enhanced yellow fluorescent marker gene in Toxoplasma gondii

Fluorescent protein is a useful tool for monitoring gene expression and studying biological processes of organisms including parasites. To improve the transfection efficiency and fluorescent intensity in Toxoplasma gondii, a new transient expression vector, RGES, containing the tandem enhanced yellow fluorescent protein gene, EYFP-EYFP, under the control of the parasite dense granule protein 1 (GRA1) promoter was constructed. The RGES plasmid was introduced into T. gondii RH strain by electroporation. A high proportion of tachyzoites (approximately 25.5%) was transfected with the EYFP-EYFP gene and high fluorescence intensity was detected 48-72 h after electroporation. The tandem EYFP gene and its expression in the transfectants were confirmed by polymerase chain reaction (PCR), reverse transcriptase PCR, and Western blotting. The efficiency of transfection and fluorescent intensity of EYFP-EYFP were greater than that of the single EYFP or green fluorescent protein. The EYFP-EYFP gene is a better marker for the study of biological processes of T. gondii.