Clonal conditional mutagenesis in malaria parasites.
We describe here an efficient method for conditional gene inactivation in malaria parasites that uses the Flp/FRT site-specific recombination system of yeast. The method, developed in Plasmodium berghei, consists of inserting FRT sites in the chromosomal locus of interest in a parasite clone expressing the Flp recombinase via a developmental stage-specific promoter. Using promoters active in mosquito midgut sporozoites or salivary gland sporozoites to drive expression of Flp or its thermolabile variant, FlpL, we show that excision of the DNA flanked by FRT sites occurs efficiently at the stage of interest and at undetectable levels in prior stages. We applied this technique to conditionally silence MSP1, a gene essential for merozoite invasion of erythrocytes. Silencing MSP1 in sporozoites impaired subsequent merozoite formation in the liver. Therefore, MSP1 plays a dual role in the parasite life cycle, acting both in liver and erythrocytic parasite stages.
Combe
Audrey
A
Institut Pasteur, Unité de Biologie et Génétique du Paludisme, 28 rue du Dr Roux, Paris cedex 15, Paris 75724, France.
Giovannini
Donatella
D
Carvalho
Teresa Gil
TG
Spath
Stephan
S
Boisson
Bertrand
B
Loussert
Céline
C
Thiberge
Sabine
S
Lacroix
Céline
C
Gueirard
Pascale
P
Ménard
Robert
R
D000818:Animals; D004254:DNA Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism; D017353:Gene Deletion; D008967:Molecular Biology / Q000379:methods*; D016296:Mutagenesis; D010962:Plasmodium berghei / Q000235:genetics*; D011995:Recombination, Genetic
D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase