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.