Journal article
Yeast dihydroorotate dehydrogenase as a new selectable marker for Plasmodium falciparum transfection
Molecular and biochemical parasitology, v 177(1), pp 29-34
May 2011
PMID: 21251930
Featured in Collection : UN Sustainable Development Goals @ Drexel
Abstract
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► yDHODH is an alternative selectable marker in Plasmodium falciparum transfections. ► Atovaquone and DSM-1 are selection agents for use with the yDHODH vector pUF-1. ► PfVP2 and CSP genes were successfully knocked out using pUF-1.
Genetic manipulation of Plasmodium falciparum in culture through transfection has provided numerous insights into the molecular and cell biology of this parasite. The procedure is rather cumbersome, and is limited by the number of drug-resistant markers that can be used for selecting transfected parasites. Here we report a new selectable marker that could allow multiple transfections. We have taken advantage of our finding that a critical function of the mitochondrial electron transport chain (mtETC) in the erythrocytic stages of P. falciparum is the regeneration of ubiquinone as co-substrate of dihydroorotate dehydrogenase (DHODH), and that transgenic P. falciparum expressing ubiquinone-independent DHODH from yeast (yDHODH) are resistant to all mtETC inhibitors. We assessed the possibility of using yDHODH as a positive selectable marker for transfections of P. falciparum, including its use in gene disruption strategies. We constructed a transfection vector designed for gene disruption, termed pUF-1, containing the yDHODH gene as the positive selection marker in combination with a previously described fused yeast cytosine deaminase-uracil phosphoribosyl transferase gene as a negative selection marker. Transfection of the D10 strain followed by selection with atovaquone yielded positively selected parasites containing the plasmid, demonstrating that yDHODH can be used as a selective marker. Atovaquone, however, could not be used for such selection with the Dd2 strain of P. falciparum. On the other hand, we demonstrated that yDHODH transgenic parasites could be selected in both strains by Plasmodium DHODH-specific triazolopyrimidine-based inhibitors. Thus, selection with DHODH inhibitors was superior in that it successfully selected transgenic Dd2 parasites, as well as yielded transgenic parasites after a shorter period of selection. As a proof of concept, we have successfully disrupted the type II vacuolar proton-pumping pyrophosphatase gene (PfVP2) in P. falciparum by double crossover recombination, showing that this gene is not essential for the survival of blood stage parasites.
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Details
- Title
- Yeast dihydroorotate dehydrogenase as a new selectable marker for Plasmodium falciparum transfection
- Creators
- Suresh M Ganesan - Center for Molecular Parasitology, Department of Microbiology and Immunology, Drexel University College of Medicine, 2900 W. Queen Lane, Philadelphia, PA 19129, USAJoanne M Morrisey - Center for Molecular Parasitology, Department of Microbiology and Immunology, Drexel University College of Medicine, 2900 W. Queen Lane, Philadelphia, PA 19129, USAHangjun Ke - Center for Molecular Parasitology, Department of Microbiology and Immunology, Drexel University College of Medicine, 2900 W. Queen Lane, Philadelphia, PA 19129, USAHeather J Painter - Center for Molecular Parasitology, Department of Microbiology and Immunology, Drexel University College of Medicine, 2900 W. Queen Lane, Philadelphia, PA 19129, USAKamal Laroiya - Center for Molecular Parasitology, Department of Microbiology and Immunology, Drexel University College of Medicine, 2900 W. Queen Lane, Philadelphia, PA 19129, USAMargaret A Phillips - Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USAPradipsinh K Rathod - Department of Chemistry, University of Washington, Seattle, WA 98195, USAMichael W Mather - Center for Molecular Parasitology, Department of Microbiology and Immunology, Drexel University College of Medicine, 2900 W. Queen Lane, Philadelphia, PA 19129, USAAkhil B Vaidya - Center for Molecular Parasitology, Department of Microbiology and Immunology, Drexel University College of Medicine, 2900 W. Queen Lane, Philadelphia, PA 19129, USA
- Publication Details
- Molecular and biochemical parasitology, v 177(1), pp 29-34
- Publisher
- Elsevier
- Resource Type
- Journal article
- Language
- English
- Academic Unit
- Microbiology and Immunology
- Web of Science ID
- WOS:000289338000004
- Scopus ID
- 2-s2.0-79952453391
- Other Identifier
- 991014878388804721
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- Collaboration types
- Domestic collaboration
- Web of Science research areas
- Biochemistry & Molecular Biology
- Parasitology