GIMO Transfection in Rodent Malaria Parasites Figure 4. Generation of a P. yoelii reporter line, PyGFP-luccon that is marker-free and expresses a fusion protein of GFP and luciferase. (A) Schematic representation of the introduction of a gfp-luciferase-expression cassette into the GIMOPy17X mother line. Construct pL1847 containing the eef1a-gfp::luciferase-39pbdhfr cassette is integrated into the modified P. yoelii 230p locus containing the hdhfr::yfcu selectable marker cassette (black box) by double cross-over homologous recombination at the target regions (hatched boxes). Negative selection with 5-FC results in selection of parasites that have the gfp-luciferase reporter introduced into the genome and the hdhfr::yfcu marker removed. Location of primers used for PCR analysis and sizes of PCR products are shown (see Table S2 for primer sequences). (B) Fluorescence microscopy of a live schizont of PyGFP- luccon; bright field (BF), DNA staining (Hoechst; Blue) and GFP expression (green). (C) PFG-separated chromosomal Southern analysis of two independent GIMO transfection parasite lines (exp. 1970 and 1971). Hybridization performed with a mixture of two probes, one specific for pb25 (chromosome 5) and the other for hdhfr (chromosome 3), demonstrating the efficiency of selection of hdhfr::yfcu ‘marker-free’ parasites in the different experiments. (D) Analysis of luciferase-expression of blood stages of 3 clones of PyGFP-luccon (exp. 1971). Luciferase-activity was measured by real time in vivo imaging of live mice with a parasitemia of 1–3%. (E) Diagnostic PCR analysis confirming correct integration of the gfp-luciferase gene in PyGFP-luccon clones (exp. 1971): amplification of hdhfr::yfcu marker (SM, primers 4698/4699), 59 integration PCR (59 int, primers 6527/6812), 39 integration PCR (39 int, primers 6813/6528) and gfp-luc (primers 6814/6815). Primer location, product sizes are shown in A and primer sequences in Table S2. doi:10.1371/journal.pone.0029289.g004 days which is comparable to the prepatancy of mice infected with 104 wild type sporozoites. Genotype analysis of Dgr(+gr) blood stage parasites after mosquito passage and sporozoite infection, by diagnostic PCR and Southern analysis of digested genomic DNA, confirmed that gr was indeed restored (i.e. complemented) in the Dgr(+gr) parasites and no deletion mutants were present (Figure 5B). The restoration of the phenotype of Dgr parasites using a PCR-amplified construct in combination with negative selection demonstrates that GIMO transfection is a fast method for gene complementation (see also the Discussion section). In addition it is a relatively simple method, requiring only PCR- amplified DNA-constructs that can be used as the constructs do not require a drug-selectable marker cassette. Discussion Genetic modification of malaria parasites is limited by the paucity of drug-selection markers that permit selection of transformed mutants, which in turn hampers the generation of multiple genetic modifications in the same mutant. The novel GIMO-transfection method reported in this study permits the generation of mutants stably expressing heterologous proteins free of drug-selectable markers, facilitating further genetic modification of the transgenic parasites. In addition, it provides a fast and simple way for gene complementation of gene deletion/mutation mutants. We have generated reference mother lines and standard ‘knock-in’ constructs for both P. berghei ANKA and P. yoelii PLoS ONE | www.plosone.org 6 December 2011 | Volume 6 | Issue 12 | e29289