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Removal of heterologous sequences from Plasmodium falciparum mutants using FLPe-recombinase

Ben C L van Schaijk1, Martijn W Vos, Chris J Janse

  • 1Department of Medical Microbiology, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands. B.VanSchaijk@mmb.umcn.nl

Plos One
|December 15, 2010
PubMed

Insights

Researchers developed a new system to create genetically modified malaria parasites without drug-resistance genes. This advancement aids Plasmodium research and the development of live, attenuated malaria vaccines.

Area of Science:

  • Molecular Biology
  • Parasitology
  • Vaccine Development

Background:

  • Genetically modified Plasmodium parasites are crucial for gene-function studies and are explored as live attenuated vaccines.
  • Current methods for generating transgenic malaria parasites rely on drug-resistance markers, limiting further genetic manipulation.

Purpose of the Study:

  • To develop a novel method for generating transgenic malaria parasites that are free of drug-resistance genes.
  • To enable efficient removal of selectable markers from the Plasmodium falciparum genome.

Main Methods:

  • Development and implementation of an Flp-recombinase (FLP) recognition target (FRT) system for marker removal.
  • Targeting two neighboring genes (p52 and p36) using a construct with an FRT-flanked selectable marker cassette.
  • Transient transfection with a plasmid expressing a thermostable enhanced FLP-recombinase for marker excision.

Main Results:

  • Demonstrated complete and efficient removal of the introduced drug-resistance gene in Plasmodium falciparum.
  • Successfully utilized the FRT/FLP system to excise the selectable marker cassette.
  • Established a method for generating genetically modified parasites devoid of resistance markers.

Conclusions:

  • The FRT/FLP system provides a powerful tool for generating marker-free transgenic Plasmodium parasites.
  • This method facilitates sequential gene targeting and the development of genetically modified parasites for live attenuated malaria vaccines.
  • Opens new avenues for Plasmodium research and vaccine development by enabling cleaner genetic modifications.

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