Puromycin-N-acetyltransferase as a selectable marker for use in Plasmodium falciparum

de Koning-Ward, TF; Waters, AP; Crabb, BS

Author(s): de Koning-Ward, TF; Waters, AP; Crabb, BS;
Details: Publication Year 2001-10,Volume 117,Issue #2,Page 155-160
Journal Title: MOLECULAR AND BIOCHEMICAL PARASITOLOGY
Publication Type: Journal Article
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 IC50 and IC90 values increasing approximate to 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. (C) 2001 Elsevier Science B.V. All rights reserved.
Publisher: ELSEVIER SCIENCE BV
Keywords: HUMAN DIHYDROFOLATE-REDUCTASE; MALARIA PARASITES; STABLE TRANSFECTION; MAMMALIAN-CELLS; STREPTOMYCES-ALBONIGER; PAC GENE; RESISTANCE; TRANSFORMATION; DISRUPTION; EXPRESSION
Publisher's Version: https://doi.org/10.1016/S0166-6851(01)00344-9
Terms of Use/Rights Notice: Refer to copyright notice on published article.

Creation Date: 2001-10-01 12:00:00