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. 2014 Jul 31;211(1):125–129. doi: 10.1093/infdis/jiu423

Figure 1.

Figure 1.

Editing of the Plasmodium vivax dhfr-ts gene with zinc-finger nucleases (ZFNs). A, Schematic representation of the pvdhfr-ts allelic modification strategy. The pvDC2-ZFNpvdhfr plasmid expresses a P. vivax dhfr–specific pair of ZFNs separated by a 2A-peptide under control of the P. vivax calmodulin promoter (5′ cam) and the P. vivax hsp86 3′ untranslated regulatory element (3′ hsp86). In addition, the plasmid carries a 1.37-kb donor region from the 1.86-kb pvdhfr-ts coding sequence with 4 engineered mutations for pyrimethamine resistance (asterisks). Expression of the ZFN pair, which functions as an obligate heterodimer, in the transfected parasites induces a double-strand break (DSB) in the pvdhfr-ts sequence (thunderbolt). The donor sequence provided on the same plasmid provided a donor template for DSB repair via homologous recombination, thereby introducing pvdhfr mutations. The primer pair pvdhfr-5′UTR-F (forward) and pvdhfr-R1 (reverse) was used to confirm editing of the genomic pvdhfr sequence. For purposes of illustration, elements of the map are not drawn to scale. B, Schematic representation of ZFNs bound to the pvdhfr target. The left and right ZFNs consist of 4 and 5 ZFN modules, respectively, which bind to opposite strands of the DNA. Upon binding, the split FokI endonuclease moieties (physically proximal to each other by virtue of each binding neighboring DNA sequences) dimerize to form a functional nuclease that can then cleave the DNA and produce a DSB at the target site. C, P. vivax transfection protocol. Splenectomized Saimiri boliviensis were infected with P. vivax Chesson. When the parasitemia reached 0.5%, blood specimens were collected and depleted of white blood cells, using a Sepacell filter. The recovered erythrocyte samples were suspended in Cytomix, mixed with plasmid DNA, and electroporated. A splenectomized malaria-naive S. boliviensis was inoculated with the electroporated cells and subsequently treated with 3 weekly doses of 4 mg/kg pyrimethamine (Pyr) to select for ZFN-edited, Pyr-resistant parasites.