TY - JOUR
T1 - Yeast dihydroorotate dehydrogenase as a new selectable marker for Plasmodium falciparum transfection
AU - Ganesan, Suresh M.
AU - Morrisey, Joanne M.
AU - Ke, Hangjun
AU - Painter, Heather J.
AU - Laroiya, Kamal
AU - Phillips, Margaret A.
AU - Rathod, Pradipsinh K.
AU - Mather, Michael W.
AU - Vaidya, Akhil B.
N1 - Funding Information:
We thank Dr. Alan Cowman for providing the plasmid pCC4. This work was supported by the United States National Institutes of Health grants AI028398 (to ABV), U01AI075594 (to MAP and PKR), and AI053680 (to MAP and PKR). We also acknowledge support from Drexel University College of Medicine . MAP acknowledges the support of the Welch Foundation (I-1257). MAP holds the Carolyn R. Bacon Professorship in Medical Science and Education.
PY - 2011/5
Y1 - 2011/5
N2 - 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.
AB - 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.
KW - Atovaquone
KW - Gene knockout
KW - Malaria
KW - Selectable marker
KW - Triazolopyrimidine
KW - Vacuolar pyrophosphatase
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U2 - 10.1016/j.molbiopara.2011.01.004
DO - 10.1016/j.molbiopara.2011.01.004
M3 - Article
C2 - 21251930
AN - SCOPUS:79952453391
SN - 0166-6851
VL - 177
SP - 29
EP - 34
JO - Molecular and Biochemical Parasitology
JF - Molecular and Biochemical Parasitology
IS - 1
ER -