Puromycin-N-acetyltransferase as a selectable marker for use in Plasmodiumfalciparum

The limited number of selectable markers available for malaria transfection has hindered extensive manipulation of the Plasmodium falciparum genome an d subsequently thorough genetic analysis of this organism. In this paper, w e 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 IC50 and IC90 values increasing approximate to 3- and 7-fold, respectively in PAC-expressing parasites. Despite this relatively low level of resistanc e, parasite populations transfected with the PAC selectable marker and sele cted directly on puromycin emerged at the same rate post-transfection as hu man dihydrofolate reductase (hDHFR)-expressing parasites, selected independ ently with the anti-folate drug WR99210. Transfected parasites generally ma intained the PAC expression plasmid episomally at between two and six copie s per parasite. We also demonstrate by cycling transfected parasites in the presence and absence of puromycin for several weeks, that the PAC selectab le marker can be used for gene-targeting. Since the mode of action of purom ycin is distinct from other drugs currently used for the stable transfectio n of P. falciparum, the PAC selectable marker should also have applicabilit y for use in conjunction with other positive selectable markers, thereby in creasing the possibilities for more complex functional studies of this orga nism. (C) 2001 Elsevier Science B.V. All rights reserved.