and PyGFP-luccon indicates that p230 is also a suitable locus to introduce transgenes in P. yoelii. Several P. berghei reference lines exist that express reporter proteins, such as GFP and luciferase, and do not contain drug- selection markers. Most of these parasites have been obtained by FACS-sorting where GFP expression is used as the selectable marker [8,9]. However, selection of transgenic fluorescent- expressing parasites by FACS-sorting has been only reported for selecting GFP-expressing parasites and not with parasites that express other fluorescent proteins. In our hands, FACS-sorting of GFP-expressing parasites is not a highly efficient selection method as often the selected population consists of both mutant and wild type parasites. Moreover, introducing a GFP-selection cassette increases the size of the transfection construct. This limits the size of the heterologous DNA that can be cloned into these vectors as it is difficult to maintain Plasmodium transfection vectors with a size larger than 14 kb in E. coli. Therefore, in comparison with FACS- sorting, the GIMO-transfection system is a more flexible and simpler system to introduce a wide range of heterologous genes into the parasite genome with the additional advantage that GIMO transfection constructs are far smaller since a selection- marker cassette is not required. In addition to the use of FACS-sorting for the generation of marker-free P. berghei mutants a ‘marker-recycling’ method has also been employed in P. berghei [10]. Specifically, transformed parasites expressing the fusion gene hdhfr::yfcu are first selected by positive selection with pyrimethamine; subsequently negative selection with 5-FC is applied to select parasites that have lost the resistance genes. The efficiency of selection of marker-free parasites is dependent on the frequency of the loss of the hdhfr::yfcu marker from the genome by homologous recombination and excision [10]. This method has been successfully used to generate marker-free reporter lines [22], to introduce two independent genetic modifications in the same parasite lines [22–24] and for complementation [10]. However, this marker-recycling method is relatively laborious and time consuming since it involves both positive and negative selection procedures and two parasite- cloning steps, a procedure requiring at least 9 weeks to complete. Further, marker-recycling method requires at least 24 mice in order to obtain a marker-free mutant (Figure 6A), in part a consequence of essential cloning procedures [19,22]. In contrast, the generation of marker-free mutants with GIMO-transfection can be achieved in only 4 weeks and requires only 11 mice (Figure 6A). The marker-recycling transfection constructs consist of the hdhfr::yfcu drug-selectable marker cassette, a transgene expression and two targeting sequences for integration into the genome (See Figure S2A). In addition, they have two identical regions of DNA sequence that can recombine (in the parasite genome) and excise the selectable marker cassette. In contrast the GIMO-constructs contain only the two genome targeting sequences and the transgene expression cassette (see Figure S2 for a comparison of the marker-recycling and GIMO constructs). The simple structure of GIMO constructs permits the cloning of larger transgenes (the GIMO constructs are smaller as the selectable marker cassette is absent) and improves the retention of plasmids in bacteria as internally repetitive regions of AT-rich Plasmodium DNA are absent. Further, after transfection with the GIMO construct, the selection of integration mutants is improved as no episomal construct DNA is maintained in the parasites and negative selection kills parasites expressing yfcu. GIMO-transfection is dependent on the transgene-expression construct replacing the hdhfr::yfcu selection cassette present in the mother line genome and the efficiency of the drug 5-FC to kill all parasites where this integration has not occurred and that are still GIMO Transfection in Rodent Malaria Parasites expressing yFCU. Interestingly, in both P. berghei and P. yoelii GIMO-transfection experiments we always observed that popula- tions of 5-FC selected parasites contain (low numbers of) parasites that still have the hdhfr::yfcu selection cassette in their genome. Further research is required to determine whether these parasites express yFCU but are able to survive 5-FC drug treatment or if these parasites have lost expression of yFCU through the mutation of hdhfr::yfcu selectable marker cassette. Experiments in our laboratory are now focused on improving the application of negative selection to mutant parasites in mice by providing 5-FC in the drinking water, which may permit treatment with higher concentrations of 5-FC and for longer periods. Notwithstanding the presence of non-transformed parasites after selection of GIMO-transfected parasites, the high percentages of transformed parasites in the populations permit the collection of the desired mutants by cloning. Using GIMO- transfection we have already been able to successfully generate multiple marker-free lines that express a variety of heterologous proteins (unpublished data JWL and SK). GIMO-transfection was used to generate a P. yoelii GFP- luciferase reporter parasite and is the first report describing the use of negative selection with 5-FC in combination with the yFCU marker for genetic modification of this parasite species. Moreover, the PyGFP-luccon line is the first P. yoelii reporter line that is marker-free and can be easily further genetically modified. Similar P. berghei reporter lines have been used to visualize and quantify host parasite interactions in vivo [13,21,25,26], analysis of drug- susceptibility [17,27,28] and in vivo quantification of liver stage development [18,29]. In this study we demonstrate that GIMO-transfection can not only be used to introduce heterologous genes but also is a fast and simple method for gene complementation. Restoration of the wild type phenotype by gene complementation is the most optimal strategy to show that a mutant phenotype is the result of the intended deletion (or mutation) and is not due to unrelated alterations in the parasite genome [2,11]. Genetic complementa- tion has not been widely applied in Plasmodium due to difficulties in making successive genetic modifications in the same parasite, and to problems inherent in cloning full-length AT-rich Plasmodium genes into bacterial plasmid vectors [11]. Till now two methods have been used to complement gene deletion mutants in P. berghei. The first method re-introduces the wild-type gene using a construct containing hdhfr as a positive selectable marker [30,31]. The encoded protein confers resistance to WR99210, and can be used to transfect gene deletion mutants that already contain the pyrimethamine resistance markers dhfr/ts from P. berghei or the dhfr from Toxoplasma gondii (tgdhfr) [7]. However, selection with WR99210 is not straightforward because of problems with dissolving this drug and because there is a reduced sensitivity to WR99210 of parasites that already contain the dhfr/ts or tgdhfr marker [7,10] (unpublished observations CJJ). The second complementation method is based on the marker-recycling, as described above. Gene deletion mutants (containing hdhfr::yfcu) are first subjected to negative selection to select for marker-free parasites, cloned and then transfection is performed with constructs containing the gene for complementation and a drug selection cassette [10] (see Figure S3A). This method requires generally 7 weeks and 14 mice to perform (Figure 6B). In contrast, complementation with GIMO-transfection takes only 2 weeks and 1 mouse (Figure 6B). Not only is the GIMO method much faster, requiring far fewer mice, but also a big advantage is that a simple PCR amplicon containing the wild-type gene can be used for complementation as no drug selectable needs to be used in the PLoS ONE | www.plosone.org 8 December 2011 | Volume 6 | Issue 12 | e29289