Gene transfection of Toxoplasma gondii using PEI/DNA polyplexes

The purpose of this study was to explore the potential of using cationic polyethylenimine (PEI) to deliver green fluorescent protein (GFP) to protozoan parasite Toxoplasma gondii. PEI/DNA polyplexes were formed using branched PEI and pEGFP-N1 plasmid with various N/P ratios that ranged from 5 to 50. With the increment of N/P ratio, the average size of formed PEI/DNA polyplexes determined by dynamic light scattering analysis decreased from 306 to 203 nm, while the surface charge of polyplexes obtained by zeta potential measurements increased from 20.2 to 36.7 mV. Gene transfection efficiency modulated by N/P ratio was determined, indicating PEI/DNA polyplexes were capable of transfecting parasites. The maximal GFP expression was observed 8 h post-transfection using N/P ratio of 30. To demonstrate the infectivity and potential use of GFP-expressing T. gondii, transfected parasites were inoculated to the monolayer of human foreskin fibroblast (HFF) cells. GFP-expressing tachyzoites were observed in intracellular milieu of the infected HFF cells one day after the infection. After 12-day culture, the bradyzoites expressing GFP within cysts were clearly visualized extracellularly. Our results revealed that PEI can be harnessed as an effective and inexpensive reagent to construct GFP-expressing T. gondii which has potential uses such as the study of interconversion stages and antimicrobial drug screening.