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. 2020 Dec 17;10:591046. doi: 10.3389/fcimb.2020.591046

Figure 1.

Figure 1

Generation and genotyping of the chimeric Pf-PvCSP parasites. A schematic representation of the Cas9 (plasmid Leiden falciparum (pLf) 0019) and donor DNA plasmids (pLf0109) constructs used to introduce the Pvcsp chimeric (Pvcsp-chi) expression cassette into the PfNF54 p47 gene locus. The Pvcsp-chi gene contains a chimeric repeat region of VK210 and VK247 alleles and is under the control of the promoter of the Pfcsp gene. The p47 homology regions (HR1, HR2) used to introduce the donor DNA, location of primers (P), sizes of restriction fragments (HpaI and EcoRI; in red), and PCR amplicons (in black) are indicated. WT, wild type; bsd, blasticidin selectable marker (bsd); hdhfr::yfcu, in donor plasmid. (B) Diagnostic PCRs confirming the correct integration of the Pvcsp-chi expression cassette into the Pfp47 locus. Diagnostic PCR: part of Pvcsp-chi open reading frame (primers P4/P5); part of Pfp47 open reading frame (primers P6/P7); 5’-integration of the plasmid into the Pf-PvCSP-chi genome (5’-Int; primers P1/P2); LR-PCR (primers P1/P3). (C) Southern analysis of HpaI and EcoRI restricted DNA of WT, and chimeric Pf-PvCSP parasites confirms the specific integration of the Pvcsp genes into the Pfp47 gene locus. (D) Growth of asexual blood-stages of Pf-PvCSP cl3 and WT PfNF54. Parasitemia (mean and S.D of 3 independent cultures) is shown during a 3-day culture period (in the semi-automated culture system). Cultures were initiated with a parasitemia of 0.1%.