Figure 5.

Knockout vs. knock-in gene targeting analyses reveal essential functions of PPAT and DPCK during blood stages development. (A) A schematic representation of the knockout vs. knock-in gene targeting strategy in which an endogenous gene locus (light gray boxes) is targeted for deletion or complementation by knockout (KO) and knock-in (KN) plasmid constructs, respectively. The knockout and knock-in plasmid constructs (dark gray boxes) share the same 3′ fragment but have different 5′ fragments that either disrupts or complement, respectively, the coding sequence of a gene by homologous double cross-over recombination. If the gene is essential for the functions of the parasite blood stages, no KO parasites will be recovered following transfection. If the gene is accessible for genetic modification, the KN parasites will be recovered. Diagnostic WT-specific or integration-specific test amplicons are indicated by lines. 36-cycles PCR genotyping confirmed the integration of KN but the KO plasmid constructs into PPAT gene locus (B) and into DPCK gene locus (C). Integration of ppat-KN and dpck-KN plasmid constructs were confirmed using oligonucleotide primer combinations that can only amplify from the recombinant loci (5′ Test and 3′ Test) in (B,C), respectively. The WT-specific PCR reaction (WT) confirmed that the parasites recovered from transfection with ppat-KO (B) and dpck-KN (C) were resistant WT parasites.