Parasite maintenance, transfection and dilution cloning

Parasites were maintained in complete media, comprising RPMI 1640 (Invitrogen) with the following additions: 2.3 g/L sodium bicarbonate, 4 g/L dextrose, 5.957 g/L HEPES, 0.05 g/L hypoxanthine, 5 g /L Albumax II, 0.025 g/L gentamycin sulfate, 0.292 g/L L-glutamine, and 10% (vol/vol) horse serum as described previously (Moon et al., 2016). Parasites were synchronized by using gradient centrifugation with 55% nycodenz (Progen) in RPMI to enrich schizonts, followed by a two-hour incubation with 4-[7-[(dimethylamino)methyl]−2-(4-fluorphenyl)imidazo[1,2-a]pyridin-3-yl]pyrimidin-2-amine (compound 2) which inhibits parasite egress (Collins et al., 2013). Incubations in compound 2 longer than 2 hr led to degeneration of schizonts and reduction in invasive capacity.

Tightly synchronized mature schizonts were transfected as described previously using the Amaxa 4D electroporator (Lonza) and the P3 Primary cell 4D Nucleofector X Kit L (Lonza)(Moon et al., 2013). 10 μl DNA including at 20 µg repair template pDonor_p230p and 20 μg pCas9/sg_p230p plasmid was used for transfections to generate eGFP expressing lines. 10 μl DNA including 15 μg repair template and 7 μg pCas9/sg_p230p plasmid was used for transfections to integrate the eGFP expression cassette into the p230p locus with PCR repair templates. For generating tagged lines 10 μg pCas/sg_GOI plasmid and 20 μg PCR repair templates were used. After 24 hr, and at daily intervals for 5 days, the medium was replaced with fresh medium containing 100 nM pyrimethamine (Sigma). Parasites were cloned out by limiting dilution. Parasites were diluted to 0.3 parasites/100 μl and 100 μl of 2% haematocrit culture was transferred to 96 flat-bottom plates in culture medium containing 200 mM L-glutamax (Sigma). After 7 days the media was changed and 0.2% fresh blood added. On day 11 the plate was screened for plaques, in an assay modified from P. falciparum (Thomas et al., 2016). Plaque positive cultures were transferred to 24 well plates containing 1 ml media with 2% haematocrit and used for genotyping.