title: The selectable marker human dihydrofolate reductase enables sequential genetic manipulation of the Plasmodium berghei genome.
abstract: Genetic transformation of malaria parasites has been limited by the number of selectable markers available. For the rodent malaria parasite, Plasmodium berghei, only a single selection marker has been at hand, utilising the dihydrofolate reductase-thymidylate synthase gene from either P. berghei or Toxoplasma gondii to confer resistance to the anti-malarial drug pyrimethamine. Here we report the use of the human dihydrofolate reductase (hDHFR) gene as a new selectable marker, which confers resistance to the antifolate inhibitor WR99210 upon both pyrimethamine sensitive and resistant isolates of P. berghei. Transfection with circular constructs containing the hDHFR gene resulted in the generation of highly resistant parasites containing multiple copies of episomally-maintained plasmids. These parasites showed around a 1000-fold increase in resistance to WR99210 compared to the parental parasites. We were also able to generate and select transgenic parasites harbouring only a single copy of hDHFR targeted into their genome, despite the fact that these parasites showed only a fivefold increase in resistance to WR99210 compared to the parental parasites. Importantly, and for the first time with malaria parasites, the hDHFR gene could be used in conjunction with the existing pyrimethamine selectable markers. This was demonstrated by reintroducing the circumsporozoite (CS) gene into transgenic CS-knockout mutant parasites that contained the P. berghei DHFR-TS selectable marker. The development of hDHFR as a second selectable marker will greatly expand the use of transformation technology in Plasmodium, enabling more extensive genetic manipulation and thus facilitating more comprehensive studies on the biology of the malaria parasite.
authors.affiliation: Department of Parasitology, Leiden University Medical Centre, Postbox 9600, 2300 RC, Leiden, The Netherlands.
authors.forename: T F
authors.initials: TF
authors.lastname: de Koning-Ward
authors.forename: D A
authors.initials: DA
authors.lastname: Fidock
authors.forename: V
authors.initials: V
authors.lastname: Thathy
authors.forename: R
authors.initials: R
authors.lastname: Menard
authors.forename: R M
authors.initials: RM
authors.lastname: van Spaendonk
authors.forename: A P
authors.initials: AP
authors.lastname: Waters
authors.forename: C J
authors.initials: CJ
authors.lastname: Janse
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D000962:Antimalarials / Q000494:pharmacology; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004351:Drug Resistance / Q000235:genetics; D005493:Folic Acid Antagonists / Q000494:pharmacology; D005819:Genetic Markers; D018503:Genome, Protozoan; D006801:Humans; D010957:Plasmids / Q000235:genetics; D010962:Plasmodium berghei / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D015800:Protozoan Proteins / Q000235:genetics; D011739:Pyrimethamine / Q000494:pharmacology; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*; D014162:Transfection; D014227:Triazines / Q000494:pharmacology
chemical_list: D000962:Antimalarials; D017931:DNA Primers; D005493:Folic Acid Antagonists; D005819:Genetic Markers; D015800:Protozoan Proteins; D014227:Triazines; C041595:circumsporozoite protein, Protozoan; C006201:BRL 6231; D013762:Tetrahydrofolate Dehydrogenase; D011739:Pyrimethamine