GIMO Transfection in Rodent Malaria Parasites Figure 5. Gene complementation using GIMO-transfection. (A) Schematic representation of the re-introduction of the glutathione reductase (gr) gene into the gr gene deletion mutant (Dgr, 1513cl1); 1513cl1 expresses the hdhfr::yfcu selectable marker (black box). Transfection with a 2.8 kb PCR-fragment amplified from wild type genomic DNA (primers 4530/3681) containing the gr gene, as well as the 59- and 39-targeting sequences, was used to re-introduce gr gene into the Dgr mutant. Negative selection with 5-FC selects for parasites that have the gr gene re-introduced into the genome replacing the hdhfr::yfcu marker (line 1761; Dgr(+gr). Location of primers used for PCR analysis, sizes of PCR products, restriction enzyme sites and sizes of the expected fragments in Southern analysis are indicated (see Tables S1 and S2 for primer sequences). (B) Diagnostic PCR analysis and Southern analysis of restricted genomic DNA confirm correct integration of the PCR fragment and complementation in Dgr(+gr) parasites: amplification of hdhfr::yfcu marker (SM; primers 4698/4699) and gr (ORF; primers 3742/3743). Primer location, product sizes are shown in A and primer sequences in Table S2. Southern blot was hybridized with 39UTR gr probe (i.e. 39 targeting region). The localization of the restriction enzymes used and the expected size of the fragments are shown in A: wt (wild type); Dgr (gr deletion mutants); Dgr(+gr) (complemented Dgr); mp (blood stages after mosquito passage). (C) Oocyst development of Dgr and Dgr (+gr) parasites. Only small, aberrant oocysts with no signs of sporozoite formation are present in Dgr infected mosquitoes at days 10–21 after feeding. In Dgr (+gr) infected mosquitoes sporozoite-containing oocysts with wild-type morphology are visible at day 12. (E) Salivary gland sporozoites of Dgr(+gr) examined by immuno-fluorescence microscopy: bright field (BF) and anti- CS antibody staining (CS, green). doi:10.1371/journal.pone.0029289.g005 17XNL, which we will make available for the research community. In GIMO-transfection of these mother lines, transgenes are introduced in the 230p locus of both P. berghei and P. yoelii. For P. berghei ANKA it has been shown that 230p is a ‘silent’ locus [20] and different reporter lines with transgenes introduced in this locus has been generated that show wild-type progression through the complete life-cycle [8,21]. Whether 230p is also a ‘silent’ locus in P. yoelii has not been reported before. Our observations of normal development of asexual stages, mosquito development and sporozoite infectivity of the P. yoelii mother line PLoS ONE | www.plosone.org 7 December 2011 | Volume 6 | Issue 12 | e29289