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FLP/FRT-mediated conditional mutagenesis in pre-erythrocytic stages of Plasmodium berghei

Nature Protocols volume 6, pages 1412–1428 (2011)Cite this article

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Abstract

We describe here a highly efficient procedure for conditional mutagenesis in Plasmodium. The procedure uses the site-specific recombination FLP-FRT system of yeast and targets the pre-erythrocytic stages of the rodent Plasmodium parasite P. berghei, including the sporozoite stage and the subsequent liver stage. The technique consists of replacing the gene under study by an FRTed copy (i.e., flanked by FRT sites) in the erythrocytic stages of a parasite clone that expresses the flip (FLP) recombinase stage-specifically—called the 'deleter' clone. We present the available deleter clones, which express FLP at different times of the parasite life cycle, as well as the schemes and tools for constructing new deleter parasites. We also outline and discuss the various strategies for exchanging a wild-type gene with an FRTed copy and for generating conditional gene knockout or knockdown parasite clones. Finally, we detail the protocol for obtaining sporozoites that lack a protein of interest and for monitoring sporozoite-specific DNA excision and depletion of the target protein. The protocol should allow the functional analysis of any essential protein in the sporozoite, liver stage or hepatic merozoite stages of rodent Plasmodium parasites.

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Figure 1: The FLP/FRT site-specific recombination system.
Figure 2: Construction of the UIS4/FLP P. berghei deleter clone and SSR efficiency at the sporozoite stage.
Figure 3: Testing SSR efficiency mediated by the TRAP/FLPL SSR system using a GFP reporter at the CS locus.
Figure 4: Constructing a deleter clone.
Figure 5: Placing an FRT site upstream of the start codon of the target gene.
Figure 6: Placing an FRT site in an intron of a multiexon target gene.
Figure 7: Placing an FRT site downstream of the stop codon of the target gene.
Figure 8: The p3′regFRT plasmid.
Figure 9: Conditional deletion of the target locus.
Figure 10: Wild-type and recombinant TRAP and UIS4 loci in the deleter clones.
Figure 11: PCR primers for assessing the outcome of gene targeting experiments.

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Acknowledgements

We thank C. Bourgouin and I. Thiéry and the other members of the Center for Production and Infection of Anopheles of Institut Pasteur for rearing the mosquitoes. This work was performed with the financial help of the American Heart Association (to P.B.); of the 'Institut Carnot-Pasteur Maladies Infectieuses' (to J.-C.B.); and of the Institut Pasteur, the 'Agence Nationale pour la Recherche' and the Howard Hughes Medical Institute International Program (to R.M.).

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Authors and Affiliations

  1. Institut Pasteur, Unité de Biologie et Génétique du Paludisme, Paris, France

    Céline Lacroix, Donatella Giovannini, Audrey Combe, Daniel Y Bargieri, Stephan Späth, Sabine Thiberge, Teresa Gil Carvalho & Robert Ménard

  2. Department of Microbiology and Molecular Genetics, University of Medicine and Dentistry of New Jersey–New Jersey Medical School, Newark, New Jersey, USA

    Dhruv Panchal & Purnima Bhanot

  3. Institut Pasteur, Unité d'Immunologie Moléculaire des Parasites, CNRS URA2581, Paris, France

    Lina Tawk & Jean-Christophe Barale

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  1. Céline Lacroix
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Contributions

C.L. performed all transfections, contributed to the characterization of conditional clones and is the protocol expert; D.G. and A.C. constructed the deleters, and inactivated and characterized MSP1, AMA1 and RON4; S.S. characterized the AMA1 and RON4 mutant; D.P. and P.B. inactivated PKG and provided Figure 6; D.B., L.T. and J.-C.B. helped to refine the 3′ excision strategy; S.T. helped with transfections; T.G.C. demonstrated the feasibility of the clonal conditional approach; and R.M. wrote the paper.

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Correspondence to Céline Lacroix or Robert Ménard.

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Lacroix, C., Giovannini, D., Combe, A. et al. FLP/FRT-mediated conditional mutagenesis in pre-erythrocytic stages of Plasmodium berghei. Nat Protoc 6, 1412–1428 (2011). https://doi.org/10.1038/nprot.2011.363

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