Figure 3. The efficiency of GIMO-transfection to select marker- free parasites that express mCherry. (A) Percentage of mCherry- positive parasites in GIMO-transfection of GIMOPbANKA (shown in Figure 2) after negative selection. The percentage of mCherry-positive parasites in six independent transfections (1794–1799) was determined by FACS analysis (see Figure 2D) and quantitative PCR (qPCR). By qPCR the ratio of mCherry and hdhfr::yfcu marker positive parasites was determined relative to the presence of a control gene hsp70, using the 22DDCT method (primers used in qPCR are described in Table S2). (B) Efficiency of selection of hdhfr::yfcu marker-free determined by Southern analysis of PFG-separated chromosomes. Hybridization performed using a mixture of two probes, one specific for pb25 (chromosome 5) and one for hdhfr (chromosome 3) showing the efficiency of selecting hdhfr::yfcu marker-free parasites in the different experiments. doi:10.1371/journal.pone.0029289.g003 GIMOPy17X mother line by double cross-over homologous recombination. Plasmid pL1847 contains a fusion gene of gfp and luciferase under the control of the P. berghei eef1a promoter (Figure 4A). Integration of this construct will result in the introduction of the gfp-luc expression cassette and a simultaneous removal of the hdhfr::yfcu gene from GIMOPy17X (Figure 4A). Transfection of GIMOPy17X parasites and negative selection was performed as described above for GIMOPbANKA. Comparable to results obtained with the transfection of GIMOPbANKA, two mice (exp. 1970 & 1971) that were infected with GIMOPy17X transfected parasites became positive at day 6 (parasitemia 1– 2%) after selection with the drug 5-FC. Analysis by fluorescence microscopy showed that ,30% and ,70% of the parasites of line 1970 and 1971, respectively, were GFP positive (Figure 4B). Southern analysis of PFG-separated chromosomes confirmed that most drug-selected parasites of line 1971 had removed the hdhfr::yfcu selectable marker (Figure 4C). We obtained three clones of line 1971 and all three expressed luciferase as shown by in vivo imaging of mice infected with 1971cl1-3 blood stages parasites GIMO Transfection in Rodent Malaria Parasites (Figure 4D). PCR analysis confirmed the correct integration of the fusion gene gfp-luciferase and removal of hdhfr::yfcu (Figure 4E). The results demonstrate that GIMO-transfection and the negative selection procedure can be applied to P. yoelii in order to generate parasites that express transgenes and are free of drug-selectable markers. In addition, these marker-free P. yoelii 1971 cloned lines (PyGFP-luccon), are excellent tools to quantitatively analyse P. yoelii development in blood and liver stages using both in vivo and in vitro luminescent assays as has been achieved with P. berghei reporter parasites [17,18]. GIMO-transfection is a rapid and simple method for gene complementation Gene complementation is used to prove that the phenotype of a gene deletion/modified parasite is the direct result of the gene mutation and not a consequence of an unintended alteration of the parasites genome [11]. Complementation is performed by reintroduction of a wild-type copy of the gene into the genome of a mutant in order to restore the wild-type phenotype, thereby establishing the association of the phenotype to the deletion genotype. We analysed whether GIMO-transfection can be used for gene complementation using a published gene deletion mutant of P. berghei with a defined phenotype. Complementation of a mutant using GIMO-transfection requires that the mutant contain the negative selectable marker yfcu in its genome. We therefore choose to complement a P. berghei mutant (Dgr) which lacks expression of glutathione reductase [19]. In this mutant, the glutathione reductase (gr) has been deleted using a construct containing the hdhfr::yfcu marker and the mutant becomes arrested in the mosquito during oocyst development with a complete absence of sporozoite production [19]. For comple- mentation of the Dgr mutant we generated a restoration DNA- construct by simply amplifying the gr gene from wild-type P. berghei genomic DNA and therefore avoided any cloning steps. Using the same primers that amplified the 59 and 39 targeting regions for the DNA construct used to generate the Dgr gene deletion mutant [19] (See Table S1), specifically the forward primer of 59 targeting region and reverse primer of 39 targeting region, a 2.8 kb PCR product that contained the complete gr gene and both targeting regions was amplified by a high fidelity proof reading polymerase (see Figure 5A). This PCR product was used to transfect Dgr parasites, with the aim to introduce the complete gr gene (‘gene insertion’) and thereby replacing the deleted gr locus, containing the hdhfr::yfcu (‘marker out’) as shown in Figure 5A. Selection of transfected parasites, using negative selection was as described above for other GIMO-transfections, and resulted in the selection of parasites (exp. 1761; Dgr(+gr)) in which the deleted gr had been replaced by the wild-type gr gene as confirmed by both diagnostic PCR and Southern analysis of digested genomic DNA (Figure 5B). We next analysed the phenotype of the complemented Dgr(+gr) parasites by comparing oocyst and sporozoite development of Dgr(+gr) and Dgr parasites in Anopheles stephensi mosquitoes. As previously reported [19], Dgr produced oocysts that abort development resulting in small degenerated oocysts without any signs of sporoblast or sporozoite formation (Figure 5C) at day 12 post infection (p.i.). The Dgr infected mosquitoes are not able to infect naive mice at day 21 p.i. In contrast, the complemented Dgr(+gr) have normal development in mosquitoes producing normal sized mature oocysts, which contain sporozoites at day 12 p.i. and salivary glands contained sporozoites at day 21 p.i. (Figure 5D). The Dgr(+gr) sporozoites are infectious as shown by injection of 104 salivary gland sporozoites in two na¨ıve Swiss mice. Both mice developed a blood stage infection with a prepatency period of 5 PLoS ONE | www.plosone.org 5 December 2011 | Volume 6 | Issue 12 | e29289