Related Experiment Video
Updated: Aug 4, 2026
Development of a Negative Selectable Marker for Entamoeba histolytica
Published on: December 12, 2010
Puromycin-N-acetyltransferase as a selectable marker for use in Plasmodium falciparum
T F de Koning-Ward1, A P Waters, B S Crabb
1The Walter and Eliza Hall Institute of Medical Research, PO Royal Melbourne Hospital, Melbourne, Vic. 3050, Australia.
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.
Insights
Researchers developed a new selectable marker for malaria parasite (Plasmodium falciparum) genetic studies. The puromycin-N-acetyltransferase (PAC) gene provides resistance to puromycin, enabling gene targeting and complex functional analyses.
Area of Science:
- Molecular Biology
- Parasitology
- Genetics
Background:
- Limited selectable markers in Plasmodium falciparum hinder genetic manipulation and analysis.
- Extensive genetic studies are crucial for understanding malaria parasites.
Purpose of the Study:
- To introduce and validate a novel selectable marker for Plasmodium falciparum transfection.
- To assess the utility of the puromycin-N-acetyltransferase (PAC) gene for genetic manipulation.
Main Methods:
- Transfection of Plasmodium falciparum with the PAC gene.
- Drug sensitivity assays using puromycin.
- Assessment of plasmid episomal maintenance.
- Gene targeting validation through drug cycling.
Main Results:
- Plasmodium falciparum exhibits sensitivity to puromycin.
- Transgenic PAC expression confers a 3- to 7-fold increase in resistance.
- PAC-expressing parasites selected on puromycin emerged at rates comparable to hDHFR-expressing parasites.
- The PAC marker facilitates gene targeting in transfected parasites.
- PAC expression plasmids were maintained episomally at 2-6 copies per parasite.
Conclusions:
- The PAC selectable marker is effective for Plasmodium falciparum transfection and genetic studies.
- Its distinct mode of action allows for use with other selectable markers, enabling complex functional analyses.
- This expands the toolkit for malaria parasite genetic research.
