A Novel ‘Gene Insertion/Marker Out’ (GIMO) Method for Transgene Expression and Gene Complementation in Rodent Malaria Parasites Jing-wen Lin., Takeshi Annoura., Mohammed Sajid, Se´ verine Chevalley-Maurel, Jai Ramesar, Onny Klop, Blandine M. D. Franke-Fayard, Chris J. Janse, Shahid M. Khan* Leiden Malaria Research Group, Department of Parasitology, Leiden University Medical Centre, Leiden, The Netherlands Abstract Research on the biology of malaria parasites has greatly benefited from the application of reverse genetic technologies, in particular through the analysis of gene deletion mutants and studies on transgenic parasites that express heterologous or mutated proteins. However, transfection in Plasmodium is limited by the paucity of drug-selectable markers that hampers subsequent genetic modification of the same mutant. We report the development of a novel ‘gene insertion/marker out’ (GIMO) method for two rodent malaria parasites, which uses negative selection to rapidly generate transgenic mutants ready for subsequent modifications. We have created reference mother lines for both P. berghei ANKA and P. yoelii 17XNL that serve as recipient parasites for GIMO-transfection. Compared to existing protocols GIMO-transfection greatly simplifies and speeds up the generation of mutants expressing heterologous proteins, free of drug-resistance genes, and requires far fewer laboratory animals. In addition we demonstrate that GIMO-transfection is also a simple and fast method for genetic complementation of mutants with a gene deletion or mutation. The implementation of GIMO-transfection procedures should greatly enhance Plasmodium reverse-genetic research. Citation: Lin J-w, Annoura T, Sajid M, Chevalley-Maurel S, Ramesar J, et al. (2011) A Novel ‘Gene Insertion/Marker Out’ (GIMO) Method for Transgene Expression and Gene Complementation in Rodent Malaria Parasites. PLoS ONE 6(12): e29289. doi:10.1371/journal.pone.0029289 Editor: Stefan Kappe, Seattle Biomedical Research Institute, University of Washington, United States of America Received October 12, 2011; Accepted November 24, 2011; Published December 27, 2011 Copyright: 2011 Lin et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by a Gates Foundation Grand Challenges Explorations Grant (to SMK; KHAN OPP1008310). JWL is supported by the China Scholarship Council-Leiden University Joint Program, and CJJ by a grant of the European Community’s Seventh Framework Programme (FP7/2007–2013) under grant agreement no. 242095. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: s.m.khan@lumc.nl . These authors contributed equally to this work. Introduction Reverse genetic technologies have been widely applied to gain an understanding of the function of genes in Plasmodium and to provide insight into the biology of malaria parasites and interactions with their hosts (for reviews see [1–3]. The availability of efficient genetic modification technologies for the rodent malaria parasites P. berghei and P. yoelii and the possibilities for analysis of these parasites throughout the complete life cycle have made P. berghei and P. yoelii the most frequently used models for analysis of gene function [2]. Targeted disruption or mutation of genes coupled with protein tagging has provided insight into Plasmodium gene function and parasite protein expression, localization and transport. Reverse genetics is not only applied to understand Plasmodium gene function by gene deletion but is also increasingly being used to generate parasites that express heterologous proteins, for example parasites having transgenes introduced into their genome to encode fluorescent or luminescent reporter proteins. Such reporter parasites have been instrumental in the visualization and analysis of parasite-host interactions in real-time in vitro and in vivo [4–6]. The use of mutant parasites to investigate host-parasite interactions as well as parasite gene function requires genetic modification systems that are flexible and easy to perform. The application of reverse genetics in P. berghei and P. yoelii is however restricted by the limited number of drug resistance genes (permitting the selection of transformed parasites) that are currently available. This low number of selection markers hampers and slows down successive modifications in the genome of the same parasite line. Currently only two resistance gene/drug combinations exist for use in rodent malaria parasites that can be used in successive transfections, specifically dhfr-ts/pyrimethamine and hdhfr/WR99210 [7]. Since both drug-selection markers confer resistance against pyrimethamine, the introduction of consecutive genetic modifications in the same parasite can only be performed by first selecting with pyrimethamine followed by WR99210 selection [7]. In order to circumvent the problem of limited drug- selection markers, GFP has been utilized as a selection marker and permits the selection of transformed P. berghei parasites by flow cytometry [8,9]. In addition, a method has been developed for removing drug-selection markers from transformed P. berghei parasites by utilizing the yeast fcu (yfcu) selection marker and negative selection with the drug 5-fluorocytosine (5-FC) [10], which kills all parasites expressing yfcu. In this method 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 for marker-free parasites that have ‘spontaneously’ lost the hdhfr::yfcu marker from their genome, achieved by a homologous recombination/excision event PLoS ONE | www.plosone.org 1 December 2011 | Volume 6 | Issue 12 | e29289