genotype analysis using standard methods of diagnostic PCR and Southern analysis of PFG-separated chromosomes [14]. Generation of a constitutively GFP-luciferase expressing P. yoelii (PyGFP-luccon) reporter line using GIMO- transfection A construct (pL1847) for GIMO-transfection in the GIMOPy17X mother line was generated by cloning an PCR-amplified GFP- luciferase expression cassette into the XmaI site of the basic P. yoelii 230p targeting construct pL1849 (see above). The GFP-luciferase expression cassette (59 eef1a-gfp::luciferase-39pbdhfr) was amplified from pL1603 (MRA-852, www.mr4.org) using primers 6599 and 6600. Transfection of GIMOPy17X parasites and negative selection of transformed parasites was performed as described above for transfection of GIMOPbANKA. Transformed parasites were collected for genotype analyses using standard methods of diagnostic PCR and Southern analysis of PFG-separated chro- mosomes [14]. Cloned parasites were analysed for luciferase expression using the in vivo imaging technology described below. Gene complementation using GIMO transfection Gene complementation was performed using the published glutathione reductase deletion mutant (Dgr) of P. berghei [19]. In this mutant (Dgr4; 1531cl1) the glutathione reductase (gr) gene has been deleted by a replacement construct (pL1538) that contains the postive-negative hdhfr::yfcu selectable marker cassette [19]. The pL1538 construct contains 59 and 39 targeting regions of gr. We used two of the primers that have been used to generate the replacement construct pL1538 to amplify gr gene from P. berghei genomic DNA using a proof reading polymerase (PhusionH, Finnzymes, Espoo, Finland). These primers (4049; forward primer for 59 targeting region and 3681; reverse primer for 39 targeting region) amplify the complete gr gene including the 59 and 39 targeting regions (see Table S1 for primer sequences). PCR resulted in amplification of a 2.8 kb fragment which was used to transfect Dgr parasites using standard transfection procedures [14]. Transformed parasites were selected by negative selection as describe above. Transformed parasites were collected for genotype analyses using standard methods of diagnostic PCR and Southern analysis of digested genomic DNA. Analysis of the phenotype of the complemented parasites, Dgr(+gr), was analysed by mosquito transmission experiments (see below). Fluorescence microscopy and FACS analysis For analysis of GFP- or mCherry- expression in blood stages of transgenic parasites, infected tail blood was collected in PBS and examined by microscopy using a Leica DMR fluorescent microscope with standard GFP and Texas Red filters. Parasites nuclei were labeled by staining with Hoechst-33258 (2 mmol/L, Sigma, NL). Images were recorded with the digital camera CoolSNAP HQ2 (Photometrics, NL) and processed with the ColourProc software [35]. The percentage of blood stages parasites that express mCherry was determined by FACS analysis of cultured blood stages. In brief, infected tail blood (10 mL) with a parastemia between 0.5 and 1% was cultured overnight in 1 mL complete RPMI1640 culture medium at 37uC under standard conditions for the culture of P. berghei blood stages [36]. Cultured blood samples were then collected and stained with Hoechst- 33258 (2 mmol/L, Sigma, NL) for 1 hr at 37uC in the dark and analysed using a FACScan (BD LSR II, Becton Dickinson, CA, USA) with filter 440/40 for Hoechst signals and filter 610/20 for mCherry fluorescence. For FACS analysis the population of GIMO Transfection in Rodent Malaria Parasites mature schizonts were selected based on the their Hoechst- fluorescence intensity [37]; see gate P2 in the left panel of Figure 2D. The percentage of mCherry-expressing parasites was calculated by dividing the number of mCherry-positive schizonts (gate P3 in right panel of Figure 2D) by the total number of schizonts (gate P2). Quantitative real-time PCR (qPCR) analysis of transformed parasites Genomic DNA extracted from blood stage parasites was used for qPCR analysis. To determine the ratio of transformed/non- transformed parasites in the selected parasite populations, PCR amplifications of the mCherry gene (only present in transformed parasites) and the hdhfr::yfcu selectable marker (only present in non-transformed) were carried out using the QuantiTect SYBR Green PCR Kit (Qiagen, Hilden, Germany) on a CFX96 thermal cycler (Bio-Rad Laboratories, The Netherlands). The housekeep- ing gene, P. berghei hsp70, was used as reference (see Table S2 for primers used). Real-time PCR cycle thresholds (CT) were calculated as the average of triplicate analyses (per genomic DNA from transgenic parasite). The ratio between mCherry and hdhfr::yfcu was calculated by the 22DDCT method relative to hsp70 [16]. The amplification efficiencies of mCherry and hsp70 did not violate assumptions of the DDCT method (data not shown). Real time in vivo imaging of the PyGFP-luccon reporter parasites in whole bodies of live mice Expression of luciferase and imaging of distribution of luciferase-expressing PyGFP-luccon parasites in whole bodies of live mice was determined by measuring bioluminescent activity using the IVIS100 in vivo imaging system (Caliper Life Sciences, USA) as described previously [21,38]. Bioluminescence of blood stage parasites was imaged in Swiss mice with asynchronous infections of PyGFP-luccon parasites at a parasitemia of 0.5–2%. Analysis of the phenotype of Dgr and complemented Dgr(+gr) parasites during mosquito transmission Infection of Anopheles stephensi mosquitoes with Dgr and Dgr(+gr) parasites as well as determination of production of oocysts and salivary gland sporozoites was performed as previously described [39]. Infectivity of sporozoites was tested by intravenous injection of Swiss OF1 mice with 104 hand dissected salivary gland sporozoites. The prepatent period was determined by light microscopy analysis of Giemsa-stained thin smears of tail blood. Prepatency (measured in days after sporozoite inoculation) is defined as the day when parasitemia reaches 0.5–2%. Indirect Immunofluorescence assay 104 Dgr(+gr) salivary gland sporozoites in 10 mL were allowed to adhere to polylysine coating slides, fixed for 15 minutes with 4% PFA, and washed 365 minutes with PBS. Sporozoites were then permeabilized with 0.5% Triton-X100 for 15 minutes followed by a 365 minutes wash with PBS. Slides were blocked 30 minutes at room temperature in 10% FCS and incubated over night with polyclonal rabbit anti-CS antiserum [40] (dilution 1:1000, kindly provided by Dr M. Yuda) at 4uC. Slides were washed 365 minutes in PBS and incubated with donkey anti-rabbit, Alexa 488-conjugated secondary antibody (dilution 1:500), 1 hr in room temperature. Slides were washed 365 minutes in PBS, and then incubated 15 minutes with Hoechst 33342 in room temperature. Prior to mounting, slides were washed for 5 minutes and analysed with were analyzed using a Leica DMR fluorescence microscope at 10006 magnification. PLoS ONE | www.plosone.org 11 December 2011 | Volume 6 | Issue 12 | e29289