title: A rapid and robust selection procedure for generating drug-selectable marker-free recombinant malaria parasites.
abstract: Experimental genetics have been widely used to explore the biology of the malaria parasites. The rodent parasites Plasmodium berghei and less frequently P. yoelii are commonly utilised, as their complete life cycle can be reproduced in the laboratory and because they are genetically tractable via homologous recombination. However, due to the limited number of drug-selectable markers, multiple modifications of the parasite genome are difficult to achieve and require large numbers of mice. Here we describe a novel strategy that combines positive-negative drug selection and flow cytometry-assisted sorting of fluorescent parasites for the rapid generation of drug-selectable marker-free P. berghei and P. yoelii mutant parasites expressing a GFP or a GFP-luciferase cassette, using minimal numbers of mice. We further illustrate how this new strategy facilitates phenotypic analysis of genetically modified parasites by fluorescence and bioluminescence imaging of P. berghei mutants arrested during liver stage development.
authors.affiliation: 1] Sorbonne Universités, UPMC Univ Paris 06, CR7, Centre d'Immunologie et des Maladies Infectieuses (CIMI-Paris), 75013, Paris, France [2] INSERM, U1135, CIMI-Paris, 75013, Paris, France [3] CNRS, ERL 8255, CIMI-Paris, 75013, Paris, France [4].
authors.forename: Giulia
authors.initials: G
authors.lastname: Manzoni
authors.affiliation: 1] Sorbonne Universités, UPMC Univ Paris 06, CR7, Centre d'Immunologie et des Maladies Infectieuses (CIMI-Paris), 75013, Paris, France [2] INSERM, U1135, CIMI-Paris, 75013, Paris, France [3] CNRS, ERL 8255, CIMI-Paris, 75013, Paris, France [4].
authors.forename: Sylvie
authors.initials: S
authors.lastname: Briquet
authors.affiliation: 1] Sorbonne Universités, UPMC Univ Paris 06, CR7, Centre d'Immunologie et des Maladies Infectieuses (CIMI-Paris), 75013, Paris, France [2] INSERM, U1135, CIMI-Paris, 75013, Paris, France [3] CNRS, ERL 8255, CIMI-Paris, 75013, Paris, France.
authors.forename: Veronica
authors.initials: V
authors.lastname: Risco-Castillo
authors.affiliation: 1] Sorbonne Universités, UPMC Univ Paris 06, CR7, Centre d'Immunologie et des Maladies Infectieuses (CIMI-Paris), 75013, Paris, France [2] INSERM, U1135, CIMI-Paris, 75013, Paris, France [3] CNRS, ERL 8255, CIMI-Paris, 75013, Paris, France [4].
authors.forename: Charlotte
authors.initials: C
authors.lastname: Gaultier
authors.affiliation: 1] Sorbonne Universités, UPMC Univ Paris 06, CR7, Centre d'Immunologie et des Maladies Infectieuses (CIMI-Paris), 75013, Paris, France [2] INSERM, U1135, CIMI-Paris, 75013, Paris, France [3] CNRS, ERL 8255, CIMI-Paris, 75013, Paris, France.
authors.forename: Selma
authors.initials: S
authors.lastname: Topçu
authors.affiliation: 1] Sorbonne Universités, UPMC Univ Paris 06, CR7, Centre d'Immunologie et des Maladies Infectieuses (CIMI-Paris), 75013, Paris, France [2] INSERM, U1135, CIMI-Paris, 75013, Paris, France [3] CNRS, ERL 8255, CIMI-Paris, 75013, Paris, France.
authors.forename: Maria Larisa
authors.initials: ML
authors.lastname: Ivănescu
authors.affiliation: 1] Sorbonne Universités, UPMC Univ Paris 06, CR7, Centre d'Immunologie et des Maladies Infectieuses (CIMI-Paris), 75013, Paris, France [2] INSERM, U1135, CIMI-Paris, 75013, Paris, France [3] CNRS, ERL 8255, CIMI-Paris, 75013, Paris, France.
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Franetich
authors.affiliation: Sorbonne Universités, UPMC Univ Paris 06, Plateforme de Cytométrie en Flux CyPS, site Pitié-Salpêtrière, Paris, France.
authors.forename: Bénédicte
authors.initials: B
authors.lastname: Hoareau-Coudert
authors.affiliation: 1] Sorbonne Universités, UPMC Univ Paris 06, CR7, Centre d'Immunologie et des Maladies Infectieuses (CIMI-Paris), 75013, Paris, France [2] INSERM, U1135, CIMI-Paris, 75013, Paris, France [3] CNRS, ERL 8255, CIMI-Paris, 75013, Paris, France [4] Assistance Publique-Hôpitaux de Paris, Hôpitaux Universitaires La Pitié Salpêtrière - Charles Foix, Service de Parasitologie-Mycologie, Paris, France.
authors.forename: Dominique
authors.initials: D
authors.lastname: Mazier
authors.affiliation: 1] Sorbonne Universités, UPMC Univ Paris 06, CR7, Centre d'Immunologie et des Maladies Infectieuses (CIMI-Paris), 75013, Paris, France [2] INSERM, U1135, CIMI-Paris, 75013, Paris, France [3] CNRS, ERL 8255, CIMI-Paris, 75013, Paris, France.
authors.forename: Olivier
authors.initials: O
authors.lastname: Silvie
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D000962:Antimalarials / Q000494:pharmacology* / Q000627:therapeutic use; D004351:Drug Resistance / Q000235:genetics; D005260:Female; D015870:Gene Expression; D017930:Genes, Reporter; D005819:Genetic Markers; D006801:Humans; D008018:Life Cycle Stages; D008099:Liver / Q000187:drug effects / Q000469:parasitology; D008163:Luminescent Measurements / Q000379:methods; D008288:Malaria / Q000188:drug therapy / Q000469:parasitology*; D051379:Mice; D021261:Parasitic Sensitivity Tests / Q000379:methods*; D010961:Plasmodium / Q000187:drug effects* / Q000235:genetics* / Q000254:growth & development; D010962:Plasmodium berghei / Q000187:drug effects / Q000235:genetics; D011995:Recombination, Genetic
chemical_list: D000962:Antimalarials; D005819:Genetic Markers
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