Selection by flow-sorting of genetically transformed, GFP-expressing blood stages of the rodent malaria parasite, Plasmodium berghei

Authors:
Janse CJ, Franke-Fayard B, Waters AP
In:
Source: Nat Protocols
Publication Date: (2006)
Issue: 1(2): 614-623
Research Area:
Parasitology
Cells used in publication:
Plasmodium berghei
Species: unicellular
Tissue Origin:
Platform:
Nucleofector™ I/II/2b
Experiment
10^6-10^8 schizonts per transfection, 5-10 µg DNA, Nucleofector solution 88A6, program U-033
Abstract
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.