title: Puromycin-N-acetyltransferase as a selectable marker for use in Plasmodium falciparum.
abstract: The limited number of selectable markers available for malaria transfection has hindered extensive manipulation of the Plasmodium falciparum genome and subsequently thorough genetic analysis of this organism. In this paper, we demonstrate that P. falciparum is highly sensitive to the drug puromycin, but that transgenic expression of the puromycin-N-acetyltransferase (PAC) gene from Streptomyces alboninger confers resistance to this drug with the IC(50) and IC(90) values increasing approximately 3- and 7-fold, respectively in PAC-expressing parasites. Despite this relatively low level of resistance, parasite populations transfected with the PAC selectable marker and selected directly on puromycin emerged at the same rate post-transfection as human dihydrofolate reductase (hDHFR)-expressing parasites, selected independently with the anti-folate drug WR99210. Transfected parasites generally maintained the PAC expression plasmid episomally at between two and six copies per parasite. We also demonstrate by cycling transfected parasites in the presence and absence of puromycin for several weeks, that the PAC selectable marker can be used for gene-targeting. Since the mode of action of puromycin is distinct from other drugs currently used for the stable transfection of P. falciparum, the PAC selectable marker should also have applicability for use in conjunction with other positive selectable markers, thereby increasing the possibilities for more complex functional studies of this organism.
authors.affiliation: The Walter and Eliza Hall Institute of Medical Research, PO Royal Melbourne Hospital, Melbourne, Vic. 3050, Australia.
authors.forename: T F
authors.initials: TF
authors.lastname: de Koning-Ward
authors.forename: A P
authors.initials: AP
authors.lastname: Waters
authors.forename: B S
authors.initials: BS
authors.lastname: Crabb
mesh_terms: D000123:Acetyltransferases / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D000962:Antimalarials / Q000494:pharmacology*; D004351:Drug Resistance; D005819:Genetic Markers; D021261:Parasitic Sensitivity Tests; D010957:Plasmids; D010963:Plasmodium falciparum / Q000187:drug effects* / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D011691:Puromycin / Q000494:pharmacology*; D013302:Streptomyces / Q000201:enzymology / Q000235:genetics; D014162:Transfection; D014227:Triazines / Q000494:pharmacology
chemical_list: D000962:Antimalarials; D005819:Genetic Markers; D014227:Triazines; C006201:BRL 6231; D011691:Puromycin; D000123:Acetyltransferases; C045336:puromycin N-acetyltransferase