Fig. 2: Generation of Luc-expressing heme pathway KO parasites, in vivo bioluminescence imaging of infected mice and genetic complementation. | Nature Communications

Fig. 2: Generation of Luc-expressing heme pathway KO parasites, in vivo bioluminescence imaging of infected mice and genetic complementation.

From: Malaria parasite heme biosynthesis promotes and griseofulvin protects against cerebral malaria in mice

Fig. 2: Generation of Luc-expressing heme pathway KO parasites, in vivo bioluminescence imaging of infected mice and genetic complementation.The alternative text for this image may have been generated using AI.

a Double-crossover recombination strategy to generate Luc-expressing PbALASKO (PbALASKOLuc) and PbFCKO (PbFCKOLuc) parasites. Black arrows represent the position of primers used for the confirmation of site-specific integration in PbKOLuc parasites (data provided in Supplementary Fig. 1). b Genomic DNA PCR confirmation for ALAS and FC deletions in PbALASKOLuc and PbFCKOLuc parasites, respectively. Lane M: 1 kb ladder; Lane 1, 3 and 5: ALAS product (2.11 kb); Lane 2, 4 and 6: FC product (1.54 kb). n = 3 independent experiments. c RT-PCR confirmation for ALAS and FC deletions. Lane M: 1 kb ladder; Lane 1, 3 and 5: ALAS product (1.92 kb); Lane 2, 4 and 6: FC product (1.05 kb). n = 3 independent experiments. d Live GFP and m-cherry fluorescence of PbControlLuc and PbKOLuc parasites. Images were captured using 100x objective. Scale bar = 5 μm. n = 4 independent experiments. e Whole body bioluminescence imaging of PbControlLuc and PbKOLuc parasite-infected mice on day 8 post-infection. n = 3 independent experiments. f Ex vivo bioluminescence imaging of liver (Li), lungs (Lu), brain (B), heart (H) and spleen (S) of PbControlLuc- and PbKOLuc-infected mice. Enlarged images of brain are shown. g Mortality curves of mice infected with PbControlLuc (n = 5), PbALASKOLuc (n = 4) and PbFCKOLuc (n = 5) parasites (***P < 0.001, log-rank (Mantel–Cox) test). h Growth analysis of PbWT (n = 6), PbFCKO+FC (n = 6) and PbFCKOLuc (n = 5) parasites in C57BL/6 female mice. The data represent two different batches (mean ± SD; n.s not significant, Two-way ANOVA). Individual data points are shown with the respective light shaded colors. i Mortality curves of mice infected with PbWT (n = 6), PbFCKO+FC (n = 6) and PbFCKOLuc (n = 5) parasites (n.s not significant, ***P < 0.001, log-rank (Mantel–Cox) test). j RMCBS score for mice infected with PbWT (n = 6), PbFCKO+FC (n = 6) and PbFCKOLuc (n = 5) parasites on day 7/8 post-infection. PbWT and PbFCKO+FC data represent the mice succumbed to ECM (mean ± SD; n.s - not significant, ***P < 0.001, unpaired t-test; two-sided). Source data are provided as a Source Data file.

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