2. 2 Scd is not required for P. berghei blood and
mosquito stage development
To address the function of Scd, we generated Scd KO parasites by
using double crossover homologous recombination (Fig. S2A). After
recombination, the Scd ORF was replaced by the GFP and hDHFR:yFCU
expression cassettes, and GFP-expressing KO parasites were confirmed
using fluorescence microscopy (Fig. S2B). The correct gene replacement
was confirmed by diagnostic PCR, and clonal lines were obtained by
limiting dilution of the parasites. The clonal lines were further
confirmed by diagnostic PCR, and the absence of the Scd ORF was
determined by locus PCR, which amplified 4.33 kb in WT GFP and 7.16 kb
in Scd KO parasites (Fig. S2C). Furthermore, we confirmed the
insertion of the targeting cassette at the correct locus by Southern
blotting. The genomic DNA was digested with EcoR V and probed with
a 5’UTR probe, which revealed a band of 3.28 kb in KO compared to 8.4 kb
in WT GFP due to the presence of an EcoR V site in the GFP
cassette (Fig. S2D). The Scd-complemented parasite line was also
generated to check the specificity of the phenotype (Fig. S2E). For
this, a Scd expression cassette consisting of the 5’UTR, ORF, and 3’UTR
was amplified and transfected into Scd KO parasites. After
recombination, the WT locus was amplified in Scd comp parasites (Fig.
S2F). Next, we checked the effect of gene deletion on parasite growth
during asexual blood stage propagation and found a similar growth
pattern of WT GFP and KO parasites (Fig. S2G). To further understand the
role of Scd in mosquito and liver stages, we transmitted the parasites
to the mosquitoes by allowing mosquitoes to feed on infected mice.
Examination on day 14 post feeding demonstrated normal development of
oocysts and oocyst-derived sporozoites in the mosquito midgut (Fig.
S3A-D). On days 18-22 post feeding, we observed intact salivary glands
that showed a normal sporozoite load and a comparable number of salivary
gland sporozoites in KO and WT GFP parasites (Fig. S3E and F). These
results indicate that Scd is dispensable for parasite blood and mosquito
stage development.