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

Molecular and Biochemical Parasitology
T F de Koning-WardB S Crabb

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 ...Continue Reading

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Citations

Mar 9, 2004·International Journal for Parasitology·Kami Kim, Louis M Weiss
Jan 30, 2004·Molecular and Biochemical Parasitology·Annemarie v d WelAlan W Thomas
Jun 18, 2003·Experimental Parasitology·Carmen Fernandez-BecerraHernando A del Portillo
Aug 8, 2002·Molecular and Biochemical Parasitology·Ping WangJohn E Hyde
Sep 19, 2002·Drug Resistance Updates : Reviews and Commentaries in Antimicrobial and Anticancer Chemotherapy·Brendan S Crabb
Sep 6, 2003·Trends in Parasitology·Donald L GardinerKatharine R Trenholme
Jul 19, 2003·Malaria Journal·Tina S Skinner-AdamsKatharine R Trenholme
Dec 23, 2010·Pharmacogenomics·Tim AndersonDavid Fidock
Nov 23, 2006·International Journal for Parasitology·Bharath Balu, John H Adams
Sep 18, 2014·The Journal of Eukaryotic Microbiology·Wesley A J Webster, Geoffrey I McFadden
May 19, 2018·FEBS Letters·Akira SogaShinya Fukumoto
Oct 15, 2014·ChemMedChem·Shaun D FontaineAdam R Renslo
May 16, 2015·Nature Reviews. Microbiology·Tania F de Koning-WardBrendan S Crabb

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