4. 6 Determination of the Scd KO phenotype
Phenotypic analyses of the Scd KO parasites were performed as
previously described (Srivastava and Mishra, 2022). Asexual blood growth
was monitored by intravenously injecting 200 µl of blood with 0.2%
parasitemia into two groups of Swiss albino mice (n=4). Parasitemia was
determined by counting Giemsa-stained blood smears. The WT andScd KO sporozoites were isolated from the infected mosquito’s
salivary glands on days 18-22 post blood meal and counted using a
hemocytometer. For the determination of in vivo infectivity, C57BL/6
mice were either intravenously inoculated with salivary gland
sporozoites or infected by mosquito bite, and blood-stage infection was
monitored by making Giemsa-stained blood smears. Another group of mice
injected with 5,000 sporozoites was sacrificed at 36, 55, and 72 hpi,
and the livers were harvested and homogenized in TRIzol reagent
(Invitrogen, USA). For the invasion assay, HepG2 cells were infected
with salivary gland sporozoites (10,000/well) and culture fixed after 1
h and stained with anti-CSP antibody (Yoshida et al. , 1980)
before and after permeabilization as described (Rénia et al. ,
1988). The detailed EEF development assay was performed as previously
described (Srivastava and Mishra, 2022). Briefly, HepG2 cells (5.5
x104) were seeded in a 48-well plate containing
collagen-coated coverslips and incubated overnight at 37°C in a
CO2 incubator. Salivary gland sporozoites (5,000/well)
were added and incubated at 37°C, and the culture was fixed using 4%
paraformaldehyde (PFA) at different time points. For the merosome assay,
HepG2 cells (100,000/well) were seeded in a 24-well plate, infected with
salivary gland sporozoites (40,000/well), and harvested at 65 hpi to
count the merosome numbers. Merosomes were injected into Swiss mice to
check the infectivity.