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

Nat Protoc. 2006;1(2):614-23. doi: 10.1038/nprot.2006.88.

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.

MeSH terms

  • Animals
  • Flow Cytometry / methods*
  • Gene Expression Regulation
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism*
  • Malaria / parasitology
  • Mice
  • Plasmodium berghei / genetics*
  • Plasmodium berghei / metabolism*
  • Promoter Regions, Genetic
  • Protozoan Proteins / genetics
  • Protozoan Proteins / metabolism
  • Rodent Diseases / parasitology
  • Transformation, Genetic*

Substances

  • Protozoan Proteins
  • Green Fluorescent Proteins