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. 2021 Jun 28;11:669088. doi: 10.3389/fcimb.2021.669088

Figure 1.

Figure 1

PfAP2-O modification and subsequent partial knockdown leads to no discernible growth phenotype. (A) The plasmid structure and its integration of tags by homologous recombination after transfection and cloning are shown. (B) PCR analysis of genomic DNA from the recombinant parasite line confirms the integration of the plasmid and absence of wild type versions in the target gene. Combination of Oligonucleotides 3 + 1 results in the amplification of unmodified locus and the band corresponding to oligonucleotides 1 + 2 are the results of modified AP2-O gene. Note that there is no PCR fragment visible in lane 4 which would indicate residual non-modified AP2-O loci. (C) Western blot using an antiHA antibody to detect PfAP2-O-GFP expression in parasites submitted to temporary knockdown by the absence of Shield-1. Lane 1: Parasites grown in the presence of Shield-1. Lane 2: Parasites grown for two reinvasions without Shield-1. Lane 3: Parasites after re-establishment of PfAP2-O-GFP by re-addition of Shield-1 for two reinvasion cycles. As a loading control, a polyclonal antiATC (plasmodial aspartate transcarbamoylase) was used (see Supplementary Material for the full exposure). (D) Growth curves from NF54::PfAP2-O_GFP_HA_DD24 with and without 0.5 µM Shield-1 (data from triplicates).