4. 5 Generation of Scd knockout (Scd KO) and complemented (Scd comp) parasites
The Scd (Pb ANKA_1110700) targeting vector was constructed by cloning two PCR products, F3 (578 bp) and F4 (573 bp), into the pBC-GFP-hDHFR:yFCU plasmid at Xho I/Sal I andNot I/Asc I, respectively. The fragments F3 and F4 were amplified from P. berghei ANKA genomic DNA using primer pairs 1039/1056 and 1041/1042, respectively. The final vector was digested with Xho I/Asc I and transfected into P. bergheischizonts as previously described (Janse et al. , 2006). Genomic DNA was isolated from the drug-resistant GFP-expressing parasites, and correct 5’ and 3’ site-specific integration was confirmed by diagnostic PCR using primers 1410/1225 and 1215/1044, respectively. Clonal lines were obtained by limiting dilution of the parasite and again confirmed for integration by PCR. The modified genomic locus was confirmed by PCR using primers 1410/1044. For Southern blotting, parasite genomic DNA was digested with EcoR V, run on a 0.7% agarose gel, transferred to a positively charged nylon membrane (Amersham Biosciences, United Kingdom) and developed as previously described (Narwal et al. , 2022). The membrane was probed with a dioxygenin-labeled 5’ probe (DIG High Prime DNA labeling and detection kit, Roche Applied Sciences, Switzerland). To generate a Scd KO complemented parasite line, a fragment consisting of the Scd 5’UTR, ORF, and 3’UTR was amplified using primers 1410/1044 and transfected into Scd KO parasites. Parasites containing restored Scd loci were selected by negative selection using a 5-fluorocytosine (5-FC) drug (Sigma, USA) as previously described (Srivastava and Mishra, 2022). Negative selection selects parasites lacking the hDHFR:yFCU cassette. The complemented line was confirmed by the absence of GFP fluorescence and amplification of the Scd expression cassette by diagnostic PCR using primers 1410/1044.