Yeast dihydroorotate dehydrogenase as a new selectable marker for Plasmodium falciparum transfection

Suresh M. Ganesan, Joanne M. Morrisey, Hangjun Ke, Heather Jill Painter Ponton, Kamal Laroiya, Margaret A. Phillips, Pradipsinh K. Rathod, Michael W. Mather, Akhil B. Vaidya

Research output: Contribution to journalArticle

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)29-34
Number of pages6
JournalMolecular and biochemical parasitology
Volume177
Issue number1
DOIs
StatePublished - May 1 2011

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Plasmodium falciparum
Transfection
Parasites
Yeasts
Atovaquone
Genes
Electron Transport
Cytosine Deaminase
Pyrophosphatases
Ubiquinone
Plasmodium
Uracil
Transferases
dihydroorotate dehydrogenase
Genetic Recombination
Cell Biology
Protons
Regeneration
Molecular Biology
Plasmids

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Parasitology

Cite this

Ganesan, S. M., Morrisey, J. M., Ke, H., Painter Ponton, H. J., Laroiya, K., Phillips, M. A., ... Vaidya, A. B. (2011). Yeast dihydroorotate dehydrogenase as a new selectable marker for Plasmodium falciparum transfection. Molecular and biochemical parasitology, 177(1), 29-34. https://doi.org/10.1016/j.molbiopara.2011.01.004
Ganesan, Suresh M. ; Morrisey, Joanne M. ; Ke, Hangjun ; Painter Ponton, Heather Jill ; Laroiya, Kamal ; Phillips, Margaret A. ; Rathod, Pradipsinh K. ; Mather, Michael W. ; Vaidya, Akhil B. / Yeast dihydroorotate dehydrogenase as a new selectable marker for Plasmodium falciparum transfection. In: Molecular and biochemical parasitology. 2011 ; Vol. 177, No. 1. pp. 29-34.
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Ganesan, SM, Morrisey, JM, Ke, H, Painter Ponton, HJ, Laroiya, K, Phillips, MA, Rathod, PK, Mather, MW & Vaidya, AB 2011, 'Yeast dihydroorotate dehydrogenase as a new selectable marker for Plasmodium falciparum transfection', Molecular and biochemical parasitology, vol. 177, no. 1, pp. 29-34. https://doi.org/10.1016/j.molbiopara.2011.01.004

Yeast dihydroorotate dehydrogenase as a new selectable marker for Plasmodium falciparum transfection. / Ganesan, Suresh M.; Morrisey, Joanne M.; Ke, Hangjun; Painter Ponton, Heather Jill; Laroiya, Kamal; Phillips, Margaret A.; Rathod, Pradipsinh K.; Mather, Michael W.; Vaidya, Akhil B.

In: Molecular and biochemical parasitology, Vol. 177, No. 1, 01.05.2011, p. 29-34.

Research output: Contribution to journalArticle

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AU - Morrisey, Joanne M.

AU - Ke, Hangjun

AU - Painter Ponton, Heather Jill

AU - Laroiya, Kamal

AU - Phillips, Margaret A.

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AU - Mather, Michael W.

AU - Vaidya, Akhil B.

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