A genetic system to study Plasmodium falciparum protein function

Nat Methods. 2017 Apr;14(4):450-456. doi: 10.1038/nmeth.4223. Epub 2017 Mar 13.

Abstract

Current systems to study essential genes in the human malaria parasite Plasmodium falciparum are often inefficient and time intensive, and they depend on the genetic modification of the target locus, a process hindered by the low frequency of integration of episomal DNA into the genome. Here, we introduce a method, termed selection-linked integration (SLI), to rapidly select for genomic integration. SLI allowed us to functionally analyze targets at the gene and protein levels, thus permitting mislocalization of native proteins, a strategy known as knock sideways, floxing to induce diCre-based excision of genes and knocking in altered gene copies. We demonstrated the power and robustness of this approach by validating it for more than 12 targets, including eight essential ones. We also localized and inducibly inactivated Kelch13, the protein associated with artemisinin resistance. We expect this system to be widely applicable for P. falciparum and other organisms with limited genetic tractability.

MeSH terms

  • Artemisinins / pharmacology
  • Drug Resistance / drug effects
  • Drug Resistance / genetics
  • Genetic Complementation Test
  • Genetic Techniques*
  • Plasmodium falciparum / drug effects
  • Plasmodium falciparum / genetics*
  • Plasmodium falciparum / growth & development
  • Protozoan Proteins / genetics*
  • Protozoan Proteins / metabolism*
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • rab5 GTP-Binding Proteins / genetics
  • rab5 GTP-Binding Proteins / metabolism

Substances

  • Artemisinins
  • Protozoan Proteins
  • Recombinant Proteins
  • artemisinin
  • rab5 GTP-Binding Proteins