Transfection of P. knowlesi. Tightly synchronized mature schizonts were purified by centrifugation over a Nycodenz cushion. ransfections were carried out using the Amaxa 4D electroporator (Lonza) and the P3 Primary cell 4D Nucleofector X Kit L (Lonza). For each transfection, DNA (20 µg) was dissolved in 10 µL TE (10 mM Tris•HCl, 1 mM EDTA, pH 8.0), then 100 µL of supplemented P3 primary cell solution added. Approximately 5–10 µL of schizonts (~5 × 107–108) were resuspended in the DNA plus P3 primary cell solution and immediately electroporated in a 4D Nucleofector X Kit L cuvette (Lonza) using program FP158. Electroporated parasites were transferred to a 1.4-mL Eppendorf tube containing 500 µL prewarmed complete medium plus 150 µL fresh RBC, and incubated at 37 °C on a thermomixer, shaking at 650 rpm while further transfections were carried out. After 30–40 min, transfected parasites were transferred to wells of a six-well plate, each containing 4.5 mL warm complete medium.