Selection by flow-sorting of genetically transformed, GFP-expressing blood stages of the rodent malaria parasite, Plasmodium berghei.
This protocol describes a methodology for the genetic transformation of the rodent malaria parasite Plasmodium berghei and the subsequent selection of transformed parasites expressing green fluorescent protein (GFP) by flow-sorting. It provides methods for: transfection of the schizont stage with DNA constructs that contain gfp as the selectable marker; selection of fluorescent mutants by flow-sorting; and injection of flow-sorted, GFP-expressing parasites into mice and the subsequent collection of transformed parasites. The use of two different promoters for the expression of GFP is described; these two promoters require slightly different procedures for the selection of mutants. The protocol enables the collection of transformed parasites within 10-12 days after transfection. The genetic modification of P. berghei is widely used to investigate gene function in Plasmodium sp. The application of flow-sorting to the selection of transformed parasites increases the possibilities of parasite mutagenesis, by effectively expanding the range of selectable markers.
Janse
Chris J
CJ
c.j.janse@lumc.nl
Franke-Fayard
Blandine
B
Waters
Andrew P
AP
D000818:Animals; D005434:Flow Cytometry / Q000379:methods*; D005786:Gene Expression Regulation; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D008288:Malaria / Q000469:parasitology; D051379:Mice; D010962:Plasmodium berghei / Q000235:genetics* / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism; D012376:Rodent Diseases / Q000469:parasitology; D014170:Transformation, Genetic
D015800:Protozoan Proteins; D049452:Green Fluorescent Proteins