Supporting Information Figure S1 Generation of P. yoelii 230p targeting con- structs using a PCR method. A. The DNA construct (pL1805) used to generate the P. yoelii GIMO mother line was created using a modified two-step PCR method. In the first PCR reaction, 59- and 39- targeting sequences of 230p were amplified from P. yoelii 17XNL genomic DNA with the primer sets 6523/ 6524 and 6525/6526 (Table S1). Primers 6524 and 6525 have 59- extensions homologues to the hdhfr::yfcu selectable marker cassette (hatched boxes). This selectable marker cassette was excised from plasmid pL0048 digested with XhoI and NotI. Primers 6523 and 6526 have 59-terminal extensions (black boxes) for the second PCR reaction. In the second PCR reaction, the 59- and 39- targeting sequences annealed to either side of the selectable marker cassette, and the joint fragment was amplified by the external anchor-tag primers 4661/4662. Before transfection, the PCR construct was digested with Asp718I and ScaI to remove the anchor-tag and with DpnI to digest any residual pL0048 plasmid. B. The basic P. yoelii 230p targeting construct (pL1849) was generated by a modified PCR method. In the first PCR reaction, 59-and 39- targeting sequences with homologous sequences (hatched boxes) and anchor-tag sequences (black boxes) were amplified as shown in A. Oligo no. 6598 that contains the joint homologous sequences interrupted by an XmaI site (hatched boxes) was used as template for the second PCR reaction. Using the external anchor-tag primers 4661/4662, a PCR product containing both targeting sequences now with the XmaI site in the middle was amplified and subsequently cloned into TOPO TA vector resulting in construct pL1849. (EPS) Figure S2 Schematic representation of the generation of marker-free gene insertion (GI) mutants using GIMO- transfection method or using the marker-recycling method. A. Generation of marker-free gene insertion mutants expressing a gene of interest (GOI; grey box) using the standard marker-recycling method. The construct containing the hdhfr::yfcu selectable maker (black box) flanked by the recombination sequences (rc, shaded boxes) targets the 230p locus by double cross-over homologous recombination at specific target regions (hatched boxes). GI mutants are obtained after transfection, using positive selection with pyrimethamine and then cloning. Subse- quently, marker-free GI mutants are selected by negative selection using 5-FC. Only those mutants that have ‘spontaneously’ lost the hdhfr::yfcu marker from their genome, achieved by a homologous recombination/excision (see arrow), survive negative selection. B. Generation of marker-free gene mutants that express a GOI (grey box) using GIMO-transfection. The construct that contains no selectable marker cassette and targets the modified GIMO mother line 230p locus that contains the hdhfr::yfcu (black box) marker, by double cross-over homologous recombination at the target regions GIMO Transfection in Rodent Malaria Parasites (hatched boxes). Marker-free GI mutants, that have GOI expression cassette introduced into the 230p locus replacing the hdhfr::yfcu marker, are obtained by negative selection with 5-FC. (EPS) Figure S3 Schematic representation of gene comple- mentation using GIMO-transfection and the marker- recycling method. A. Gene deletion and complementation using the marker-recycling method. The gene deletion construct, containing the hdhfr::yfcu selectable maker (black box) flanked by the recombination sequences (rc; shaded boxes), targets the gene of interest (GOI) by double cross-over homologous recombination at the target regions (hatched boxes). Gene deletion mutants are obtained after transfection and positive selection with pyrimeth- amine, and cloning. Subsequently, marker-free gene deletion mutants are selected by negative selection using 5-FC. Only those mutants that have ‘spontaneously’ lost the hdhfr::yfcu marker from their genome, achieved by a homologous recombination/excision event (see arrow), survive negative selection.Complementation of the (cloned) marker-free gene deletion mutant is performed using constructs that contain a GOI expression cassette and a positive selectable marker cassette. These constructs can target either the original deleted locus or a locus that is redundant or functionally silent. Complemented parasites are selected by positive selection. B. Gene deletion and complementation using the GIMO- transfection method. The gene deletion construct containing the hdhfr::yfcu selectable maker fusion (black box) targets the GOI by double cross-over homologous recombination at specific target regions (hatched boxes). Gene deletion mutants are obtained after transfection using positive selection with pyrimethamine and then cloning. These constructs do not include recombination (rc) sequences (see A). Complementation of the gene deletion mutant is performed using a PCR fragment amplified from genomic DNA using the same outer primers used to generate the gene deletion construct (i.e. the forward primer of the 59UTR and the reverse primer of 39UTR, indicated by arrows). Integration of the PCR fragment by homologous recombination restores the deleted gene locus replacing the hdhfr::yfcu maker. Complemented parasites are selected by negative selection. (EPS) Primers used for DNA construct generation. Primers used for genotype analysis. Table S1 (DOC) Table S2 (DOC) Acknowledgments We would like to thank Dr. Stephen Hoffman for kindly providing us with P. yoelii 17XNL parasites and Dr. Masao Yuda for providing us with anti- CS antibodies. Author Contributions Conceived and designed the experiments: JWL TA CJJ SMK. Performed the experiments: JWL TA MS SC-M JR OK CJJ. Analyzed the data: JWL TA CJJ SMK. Contributed reagents/materials/analysis tools: BF-F. Wrote the paper: JWL CJJ SMK. References 1. Carvalho TG, Menard R (2005) Manipulating the Plasmodium genome. Curr Issues Mol Biol 7: 39–55. 2. Janse CJ, Kroeze H, van WA, Mededovic S, Fonager J, et al. (2011) A genotype and phenotype database of genetically modified malaria-parasites. Trends Parasitol 27: 31–39. 3. Balu B, Adams JH (2007) Advancements in transfection technologies for Plasmodium. Int J Parasitol 37: 1–10. 4. Heussler V, Doerig C (2006) In vivo imaging enters parasitology. Trends Parasitol 22: 192–195. 5. Amino R, Menard R, Frischknecht F (2005) In vivo imaging of malaria parasites–recent advances and future directions. Curr Opin Microbiol 8: 407–414. 6. Silvie O, Mota MM, Matuschewski K, Prudencio M (2008) Interactions of the malaria parasite and its mammalian host. Curr Opin Microbiol 11: 352–359. PLoS ONE | www.plosone.org 12 December 2011 | Volume 6 | Issue 12 | e29289