title: FLP/FRT-mediated conditional mutagenesis in pre-erythrocytic stages of Plasmodium berghei.
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.
authors: Institut Pasteur, Unité de Biologie et Génétique du Paludisme, Paris, France. celine.lacroix@pasteur.fr
authors: Céline
authors: C
authors: Lacroix
authors: Donatella
authors: D
authors: Giovannini
authors: Audrey
authors: A
authors: Combe
authors: Daniel Y
authors: DY
authors: Bargieri
authors: Stephan
authors: S
authors: Späth
authors: Dhruv
authors: D
authors: Panchal
authors: Lina
authors: L
authors: Tawk
authors: Sabine
authors: S
authors: Thiberge
authors: Teresa Gil
authors: TG
authors: Carvalho
authors: Jean-Christophe
authors: JC
authors: Barale
authors: Purnima
authors: P
authors: Bhanot
authors: Robert
authors: R
authors: Ménard
mesh_terms: D000818:Animals; D000852:Anopheles / Q000469:parasitology; D055786:Gene Knockout Techniques; D005818:Genetic Engineering / Q000379:methods*; D051379:Mice; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D010962:Plasmodium berghei / Q000235:genetics*; D051381:Rats; D017208:Rats, Wistar; D011995:Recombination, Genetic; D034101:Sporozoites / Q000502:physiology