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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.

SUBMITTER: Manzoni G 

PROVIDER: S-EPMC3996467 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature

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A rapid and robust selection procedure for generating drug-selectable marker-free recombinant malaria parasites.

Manzoni Giulia G   Briquet Sylvie S   Risco-Castillo Veronica V   Gaultier Charlotte C   Topçu Selma S   Ivănescu Maria Larisa ML   Franetich Jean-François JF   Hoareau-Coudert Bénédicte B   Mazier Dominique D   Silvie Olivier O  

Scientific reports 20140423


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  ...[more]

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