GIMO Transfection in Rodent Malaria Parasites Figure 1. Generation and genotype analyses of P. berghei and P. yoelii GIMO mother lines. (A) Schematic representation of the constructs used to introduce the positive-negative selectable maker cassette in the P. berghei (PbANKA) or P. yoelii (Py17XNL) 230p locus. DNA constructs pL1603 (targeting P. berghei 230p, PBANKA_030600) and pL1805 (targeting P. yoelii 230p, PY03857) containing a fusion of the positive drug selectable marker hdhfr (human dihydrofolate reductase) and negative marker yfcu (yeast cytosine deaminase and uridyl phosphoribosyl transferase) under the control of the eef1a promoter target the 230p locus at the target regions (hatched boxes) by double cross-over homologous recombination. Location of primers used for PCR analysis and sizes of PCR products are shown (see Table S2 for all primer sequences). (B) Diagnostic PCR and Southern analysis of PFG- separated chromosomes confirming correct integration of the construct in the P. berghei mother line GIMOPbANKA: 59 integration PCR (59 int; primers 5510/3189), 39 integration PCR (39 int; primers 4239/5511), amplification of hdhfr::yfcu marker (SM; primers 4698/4699) and the original P. berghei 230p (230p; primers 1637/5600). Primer location (black arrows) and product sizes are shown in A. For Southern analysis, PFG-separated chromosome were hybridized using a 39UTR pbdhfr probe that recognizes the construct integrated into P. berghei 230p locus on chromosome 3 and the endogenous locus of dhfr/ts on chromosome 7. (C) Diagnostic PCR and Southern analysis of PFG-separated chromosomes confirming correct integration of the construct in the P. yoelii mother line GIMOPy17X: 59 integration PCR (primers 6527/4770), 39 integration PCR (primers 4771/6528), amplification of hdhfr::yfcu marker (primers 4698/4699) and the P. yoelii 230p original locus (primers 6529/6530). Primer location (grey arrows) and product sizes are shown in A. For Southern analysis, chromosomal hybridization using a 39UTR pbdhfr probe recognizes the construct integrated into P. yoelii 230p locus on chromosome 3 and the endogenous locus of dhfr/ts on chromosome 7. doi:10.1371/journal.pone.0029289.g001 selected populations a low percentage of parasites still contain the hdhfr::yfcu gene (Figure 3B). These observations indicate that the application of negative selection after transfection of GIMOPbANKA, while it highly enriches for transformed parasites, it does not generate a pure population of marker-free parasites. Therefore, parasite cloning after negative selection is an essential step in GIMO-transfection in order to obtain correctly transformed parasites that express the transgene and are drug- selectable marker free. Generation of a P. yoelii reporter line, PyGFP-luccon, which is marker-free and expresses a GFP-luciferase fusion protein, by GIMO-transfection The application of negative selection to genetic modification of P. yoelii has not been reported. To test the possibility to select P. yoelii parasites lacking hdhfr::yfcu from a population of hdhfr::yfcu- containing parasites by negative selection, we generated a construct (pL1847) that targets the modified py230p locus of the PLoS ONE | www.plosone.org 3 December 2011 | Volume 6 | Issue 12 | e29289