exciting development can be partnered with GIMO transfection by ensuring all these targeting constructs have a positive-negative (hdhfr::yfcu) selectable marker cassette. Consequently all resulting mutants would be receptive to GIMO transfection thereby permitting further modification (e.g. reporter protein expression) and complementation. Materials and Methods Experimental animals and parasites Female Swiss OF1 mice (6–8 weeks old; Charles River/Janvier) were used. All animal experiments of this study were approved by the Animal Experiments Committee of the Leiden University Medical Center (DEC 07171; DEC 10099). The Dutch Experiments on Animal Act is established under European guidelines (EU directive no. 86/609/EEC regarding the Protection of Animals used for Experimental and Other Scientific Purposes). Two reference rodent malaria parasite lines were used: P. berghei ANKA line cl15cy1 [14] and P. yoelii 17XNL (clone 1.1) parasite line [32]. Generation of GIMO mother lines in P. berghei ANKA and P. yoelii 17XNL To generate the GIMO mother line in P. berghei, a DNA- construct pL1603 was generated for integration into the 230p gene (PBANKA_030600) by cloning the 59 and 39 regions of 230p as previously described [8]. The targeting sequences were amplified from genomic DNA using primer sets 5585/5586 and 5587/5588 (See Table S1 for the sequence of all primers) and cloned into the restriction sites of HindIII/KspI and Asp718I/EcoRI of the standard cloning vector pL0034 (MRA-849, www.mr4.org), which contains the hdhfr::yfcu selectable marker under the control of the eef1a promoter [10]. The hdhfr::yfcu marker is a fusion gene of the positive selection marker human dihydrofolate reductase and the negative selection marker which is a fusion gene of yeast cytosine deaminase and uridyl phosphoribosyl transferase [10]. Prior to transfec- tion the DNA-construct pL1603 was linearized with HindIII and EcoRI. To generate the GIMO mother line in P. yoelii, a modified two step PCR method [33] was used to generate DNA-construct pL1805 for integration into the 230p gene (PY03857) of P. yoelii (Figure S1A). In the first PCR reaction two fragments (59- and 39- targeting sequences, both ,1 kb) 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 (CATC- TACAAGCATCGTCGACCTC in 6524 and CCTTCAA- fTTTCGGATCCACTAG in 6525). This selectable marker cassette was excised by digestion with XhoI and NotI from a plasmid (pL0048) that contains the P. berghei eef1a-hdfhr::yfcu- 39dhfr/ts (i.e. promoter-drug selectable marker-39 terminator sequence) selection cassette. Primers 6523 and 6526 have 59- terminal extensions with an anchor-tag suitable for the second PCR reaction. In the second PCR reaction, the amplified 59- and 39- targeting sequences were annealed to either side of the selectable marker cassette, and the joint fragment was amplified by the external anchor-tag primers 4661/4662, resulting in the PCR- based targeting construct with an expected size of 4.7 kb (2.7 kb of the selectable marker cassette plus two targeting sequences of 1 kb). Before transfection, the PCR-based construct was digested with Asp718I and ScaI (in primers 6523 and 6526, respectively) to remove the ‘anchor-tag’ and with DpnI that digests any residual pL0048 plasmid. GIMO Transfection in Rodent Malaria Parasites Transfection in P. berghei ANKA and P. yoelii 17XNL, selection and cloning of the mother lines were performed by standard procedures described for transfection of P. berghei [14]. DNA- construct pL1603 was introduced into P. berghei generating mother line, GIMOPbANKA (1596cl1), and DNA construct pL1805 was introduced into P. yoelii generating mother line, GIMOPy17X (1923cl1). Correct integration of the constructs was verified by diagnostic PCR analysis (see Table S2 for primers used) and Southern blot analysis of pulse-field gel (PFG) electrophoresis- separated chromosomes probed with the 39 untranslated region (UTR) of the dhfr/ts gene of P. berghei. Generation of basic constructs without selection marker and that target the 230p locus of the GIMOPbANKA and GIMOPy17X mother lines To generate a basic P. berghei 230p-targeting construct (pL0043), the 230p targeting regions as well as the ampicillin resistance gene were amplified from plasmid pL1063 (MRA-852, www.mr4.org) using primers 5116/5117 (Table S1). A multiple cloning site (MCS) was amplified from pCRII-Blunt-TOPO vector (Zero Blunt TOPO PCR Cloning Kit, Invitrogen, Groningen, The Netherlands) using M13 forward and reverse primers. The two PCR products were digested with Asp718I and NotI restriction enzymes and ligated together creating the targeting construct pL0043. A basic P. yoelii 230p-targeting construct (pL1849) was generated using a modified 2-step PCR method (Figure S1B). In the first PCR reaction, 59-and 39- targeting sequences (both ,1 kb) of 230p were amplified from P. yoelii 17XNL genomic DNA with the primer set 6523/6534 and 6525/6526 (Table S1). As described above these primers contain 59- extensions homologues to the hdhfr::yfcu selectable marker cassette and 59-terminal extensions with an anchor-tag suitable for the second PCR reaction. A 55 nt oligo (oligo 6598; GAGGTCGACGATGCTTGTAGATGC- CCGGGCCTTCAATTTCGGATCCACTAG) containing a XmaI restriction site flanked by 2 sequences homologues to the hdhfr::yfcu selectable marker cassette was used to join the two 230p targeting regions (Figure S1B). In the second PCR reaction an fragment containing both 230p targeting sequences interrupted by the XmaI site was amplified, using the external anchor-tag primers 4661/4662, resulting in the PCR product of ,2 kb. The PCR product was cloned into TOPO TA vector (TOPO TA CloningH Kit, Invitrogen, Groningen, The Netherlands) resulting in construct pL1849. Generation of a mCherry reporter test construct and GIMO-transfection in the P. berghei mother line, GIMOPbANKA A test construct (pL1628) for GIMO-transfection in the GIMOPbANKA mother line was generated by transferring the mCherry-expression cassette (59pbeef1a-mCherry-39pbdhfr) from plas- mid pL0017-mCherry [34] into the basic 230p targeting construct pL0043 (see above) using restriction sites EcoRV/Asp718I. This plasmid was linearized with KspI before transfection. Transfection was performed as described [14]. Transformed parasites were selected by negative selection by the administration the drug 5-FC (Sigma) to mice infected with transfected parasites. Specifically; 0.4 g/kg bodyweight of 5-FC (stock: 20 mg/ml in 16PBS) administered by intra-peritoneal injection; one dose per day; for a period of 4 days, starting at 24 hours after transfection. Transformed parasites were collected at day 6/7 (infected tail blood) for phenotype analysis by fluorescence microscopy and FACS (see below) and at day 7/8 (infected heart blood) for PLoS ONE | www.plosone.org 10 December 2011 | Volume 6 | Issue 12 | e29289