around the selection cassette [10]. Both the selection of GFP- expressing mutants by flow cytometry and selection of ‘spontane- ous’ marker-free mutants by negative selection have their limitations. They are laborious and time consuming, and also require the use of many extra animals as additional cloning steps in mice are required; therefore these methods are not commonly used for successive genetic modifications or for complementation studies [11]. Here we report the development and application of a novel ‘gene insertion/marker out’ (GIMO) system for transfection of two rodent malaria parasites, P. berghei and P. yoelii. For both species we have created reference mother lines that contain the hdhfr::yfcu selection marker stably integrated into the silent 230p genomic locus. We show that transfection of these mother lines with DNA- constructs that target the modified 230p locus, followed by negative selection of transformed parasites with 5-FC is a simple and fast method to generate mutants that stably express heterologous proteins and are free of drug-selectable markers. These mother lines are therefore useful tools to generate a wide range of mutants expressing reporter and/or other heterologous proteins (under the control of different promoters) without restricting subsequent modification of the genome of these parasites. In addition, we demonstrate that GIMO-transfection is a simple and fast method to genetically complement, restoring the wild-type genotype of parasite mutants with a gene deletion or gene mutation. Importantly, GIMO transfection can be easily partnered for use with a recently developed ‘recombineering’ system for high-throughput, genome wide and highly efficient generation of gene targeting constructs [12]. Results Generation of the P. berghei and P. yoelii ‘gene insertion/ marker out’ (GIMO) mother lines For both P. berghei ANKA and P. yoelii 17XNL transgenic parasites were generated that express a fusion of a drug resistance gene and a drug sensitivity gene, the so called postive-negative selectable marker (SM), constitutively expressed by the P. berghei eef1a promoter (Figure 1A). Specifically, these parasites contain a fusion gene of hdhfr (human dihydrofolate reductase; positive SM) and yfcu (yeast cytosine deaminase and uridyl phosphoribosyl transferase; negative SM) stably integrated into the 230p locus (PBANKA_030600 in P. berghei and PY03857 in P. yoelii) through double cross-over recombination. These lines are named GIMO mother lines (gene insersion/marker out); for P. berghei GIMOPbANKA (line 1596cl1) and for P. yoelii GIMOPy17X (line 1923cl1). Both GIMO mother lines were cloned after transfection by positive selection with pyrimethamine. Correct integration of the hdhfr::yfcu selectable marker cassette in the 230p locus was demonstrated by PCR and Southern analysis of chromosomes separated by pulse-field gel electrophoresis (Figure 1B and 1C). The multiplication rate of asexual blood stages per 24 h as determined in mice infected with a single parasite [13], gametocyte production and production of oocysts and sporozoites were identical to those of the parent P. berghei and P. yoelii lines (data not shown). These GIMO mother lines are used for introduction of transgenes into the modified 230p locus through transfection with constructs that target the 230p locus. These constructs insert into the 230p locus (‘gene insertion’), thereby removing the hdhfr::yfcu selectable marker (‘marker out’) from the genome of the mother lines. Transgenic parasites that are marker-free are subsequently selected by applying negative drug selection using 5-FC (see below). GIMO Transfection in Rodent Malaria Parasites Assessing the efficiency of GIMO-transfection to select transgene expressing, drug-selectable marker-free P. berghei parasites We generated a test DNA-construct containing a transgene expression-cassette to test the efficiency of selection of transgenic mutants through the application of negative selection using 5-FC after transfection into the GIMOPbANKA mother line. This construct contains the mCherry gene under the control of the constitutive eef1a promoter and 230p targeting sequences (Figure 2A) and lacks a drug selectable marker cassette. This DNA-construct, pL1628, targets the same regions in the 230p locus in which hdhfr::yfcu selection cassette was introduced in the GIMOPbANKA mother line (Figure 2A). Transfection of GIMOPbANKA (exp. 1645) was performed using standard procedures [14] except that after transfection negative drug selection was applied instead of positive drug selection. This negative selection was performed by treating mice that were infected with transfected parasites with the drug 5-FC for 4 consecutive days (one dose per day of 10 mg), starting 24 hours after transfection. Transfected parasites of line 1645 were collected at day 7 and 8 after transfection (at a parasitemia of 0.5–3%) for phenotype and genotype analyses. Diagnostic PCR and Southern analysis of separated chromosomes confirmed the correct integration of the test construct and simultaneous removal of the hdhfr::yfcu selection cassette (Figure 2B). Analysis of mCherry expression by fluores- cence microscopy in blood stage parasites of line 1645 showed that .90% of the parasites expressed mCherry (Figure 2C). Quanti- fication of the percentage of mCherry-expressing parasites was performed by FACS analysis of mature schizonts collected from overnight blood stage cultures. Expression of transgenes, such as mCherry, under the control of the eef1a promoter increases with the maturation of parasites inside blood cells and therefore FACS quantification is improved by analysing mature schizont stages (these stages are selected based on Hoechst-fluorescence) [15]. FACS analysis confirmed that .90% (93%61.1 Figure 2D) of the schizonts were mCherry positive. Since episomal constructs cannot be maintained during selection in GIMO-transfected parasites (see Discussion), these analyses demonstrate that GIMO-transfection permits the selection parasites that express transgenes and are marker-free. To further investigate the efficiency of the GIMO system, we performed a set of independent transfections with the DNA- construct pL1628 (exp. 1794–1799) in the GIMOPbANKA mother line. In these experiments transfected parasites were selected using negative selection as described above and mCherry expression analysed by FACS (Figure 3A). In 5 out of 6 transfection experiments, the percentage of mCherry-expressing parasites was higher than 75%, whereas in one experiment (exp. 1798) 32% of schizonts were mCherry positive (Figure 3A). The presence of mCherry negative parasites in the drug-selected population indicates that non-transformed parasites survived the drug-selection but presumably still carry the hdhfr::yfcu cassette. We therefore analysed the genotype of the selected populations of all experiments by quantitative real-time PCR (qPCR) and Southern analysis of separated chromosomes to determine the ratio between parasites with and without hdhfr::yfcu. For qPCR, CT values of amplification of mCherry, hdhfr::yfcu and the control hsp70 gene were determined and the percentage of mCherry positive parasites was calculated as the relative ratio between mCherry and hdhfr::yfcu using the 22DDCT method [16]. The percentage of mCherry positive parasites based on qPCR correlated well with the percentage determined by FACS analysis (Figure 3A). Southern analysis also showed that in the PLoS ONE | www.plosone.org 2 December 2011 | Volume 6 | Issue 12 | e29289