title: Rapid positive selection of stable integrants following transfection of Plasmodium falciparum.
abstract: With the near-completion of the genome sequence of Plasmodium falciparum, further understanding of this major human pathogen urgently requires more effective genetic tools. These must include faster and more reliable gene replacement or gene knockout techniques, essential for the analysis of gene function. We describe a serial system which uses the blasticidin S deaminase (bsd) gene of Aspergillus and the neomycin phosphotransferase II (neo) gene from transposon Tn5 as selectable markers for, respectively, transient transfection of malaria parasites and the selection of stable integrants. Challenge with blasticidin S (BS) enriches the parasite population transiently expressing the bsd gene, laying the foundation for the subsequent, much less frequent, integration event. Positive selection for this rare event is enormously facilitated by fusing the neo gene in frame to the replacement or knockout targeting gene. The sequence employed for the targeting (the polymorphic pppk-dhps gene of P. falciparum, as a model system) is truncated at the 5' end with no promoter located upstream, therefore neo cannot be expressed without specifically integrating within the genomic copy of the target gene. After BS selection, the culture is immediately exposed to geneticin (G418), leading to an apparently homogenous population of mutant parasites. As well as excluding spurious integrants at non-targeted sequences, this system greatly reduces the lengthy selection period for obtaining the desired mutants by eliminating the drug-on and drug-off cycles for the production of stable integrants, which are normally required by the single marker systems currently in use for transfection of malaria parasites.
authors.affiliation: Department of Biomolecular Sciences, University of Manchester Institute of Science and Technology, PO Box 88, M60 1QD, Manchester, UK.
authors.forename: Ping
authors.initials: P
authors.lastname: Wang
authors.forename: Qi
authors.initials: Q
authors.lastname: Wang
authors.forename: Paul F G
authors.initials: PF
authors.lastname: Sims
authors.forename: John E
authors.initials: JE
authors.lastname: Hyde
mesh_terms: D000619:Aminohydrolases / Q000235:genetics; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001230:Aspergillus; D004251:DNA Transposable Elements; D018390:Gene Targeting; D005819:Genetic Markers; D005839:Gentamicins / Q000494:pharmacology; D019868:Kanamycin Kinase / Q000235:genetics / Q000378:metabolism; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism; D009705:Nucleosides / Q000494:pharmacology; D010963:Plasmodium falciparum / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D012641:Selection, Genetic; D014158:Transcription, Genetic; D014162:Transfection
chemical_list: C105176:6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase-7,8-dihydropteroate synthase; D000900:Anti-Bacterial Agents; D004251:DNA Transposable Elements; D005819:Genetic Markers; D005839:Gentamicins; D009097:Multienzyme Complexes; D009705:Nucleosides; D011993:Recombinant Fusion Proteins; C004500:blasticidin S; C010680:antibiotic G 418; D019868:Kanamycin Kinase; D000619:Aminohydrolases; C019795:blasticidin S deaminase