Transfecting Plasmids into Malaria Parasites

Both plasmids were then transfected into Dd2 P. falciparum parasites cell lines, a multi-drug resistant strain derived from an Indochina isolate (Reilly et al., 2007 (link)). Transfection was performed by electroporation in uninfected erythrocytes (Hasenkamp et al., 2012 (link)). First, erythrocytes are washed with cytomix [10 mM/L K²HPO⁴/KH²PO⁴, 120 mM/L KCl, 0.15 mM/L CaCl₂, 5 mM/L MgCl₂, 25 mM/L HEPES, 2 mM/L EGTA, adjusted with 10 M/L KOH to pH 7.6]. Afterward, 300 µL of erythrocytes were added to the plasmid solution, which contains 50 µg of plasmid and up to 200 µL of cytomix. This was transferred to a Gene Pulser/MicroPulser™ Electroporation Cuvettes, 0.2 cm gap (Bio-Rad Laboratories, Inc) and eletric shocked on the Gene Pulser Xcell™ (Bio-Rad Laboratories) electroporator. The mixture was washed twice in 5 mL of malaria culture medium (MCM) [RPMI, 1640 (Gibco) with 2 mM L-glutamine, 200 µM hypoxanthine, 0.25 µg/mL gentamycin, 25 mM HEPES, 0.2% NaHCO3, and 0.25% Albumax II (Life Technologies)] to remove lysed erythrocytes. The transfected erythrocytes were then inoculated with P. falciparum Dd2 strain parasite erythrocytes at the trophozoite stage and 5 mL of MCM.