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. 2022 Aug 24;25(9):104996. doi: 10.1016/j.isci.2022.104996

Figure 4.

Figure 4

Genetic interrogation of P. falciparum SHs

(A) Conditional allelic replacement strategy. The p2TA-cMUT construct has a homology region (HR, grey), loxPint sites (pink triangles), two differentially recodonized regions (RR1& RR2, turquoise), a triple HA tag (yellow), a T2A peptide sequence (purple), a npt selection marker (orange), gfp (green) and a hdhfr cassette (white). Upon RAP treatment, the sequence between the two loxPint sites is excised (post-excision locus). Arrows and red hexagons indicate start and stop codons, respectively. Primer binding sites are shown with dashed lines and half arrows.

(B) Diagnostic integration PCRs. Primers were design to detect the endogenous locus or the 5’ and 3’ end integration sites. PCRs were performed on gDNA from pre-clonal (mix) and 2 clonal lines. B11 gDNA was used as a negative control. For abH112-cMUT and abH114-cMUT PCR conditions to observe 5’ integration could not be obtained.

(C) Excision PCRs were performed on gDNA from DMSO- or RAP-treated schizonts or merozoites. RAP treatment results in the smaller PCR products or lack of it.

(B and C) Primer binding sites are shown in A. The expected sizes of the PCR products are shown by arrowheads.

(D) WB analysis for two clones per cMUT line was performed on lysates from DMSO- or RAP-treated schizonts or merozoites using an anti-HA antibody. RAP treatment results in the loss of HA signal.

(E)Parasite replication assay of DMSO- or RAP-treated parasites from two clonal lines per cMUT was carried out under standard conditions starting at 0.1% parasitemia. Samples were taken at 45 hpi in the first cycle, and approximately at 30 hpi in each subsequent cycle. Parasites were fixed, stained with Hoechst, and parasitemia quantified by flow cytometry. Two clones are represented in black and grey, DMSO and RAP treated samples by filled and empty circles, respectively. Data is represented as mean +/− standard deviation of three replicates.

(B–E) cMUT clones: Psta1, 1A & 1G; Psta2, 3F4 & 3E9B; abH112, 5A & 5G; abH114, 7A & 7G.