Yeast dihydroorotate dehydrogenase as a new selectable marker for Plasmodium falciparum transfection.
Yeast dihydroorotate dehydrogenase as a new selectable marker for Plasmodium falciparum transfection.
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DOI：
10.1016/j.molbiopara.2011.01.004
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发表时间：
2011-05
影响因子：
1.5
通讯作者：
Vaidya, Akhil B.
中科院分区：
文献类型：
--
作者：
Ganesan, Suresh M.;Morrisey, Joanne M.;Ke, Hangjun;Painter, Heather J.;Laroiya, Kamal;Phillips, Margaret A.;Rathod, Pradipsinh K.;Mather, Michael W.;Vaidya, Akhil B.
通过转染对培养的恶性疟原虫进行遗传操作，为这种寄生虫的分子和细胞生物学提供了许多见解。该程序相当麻烦，并且受到可用于选择转染的寄生虫的耐药标记物的数量的限制。在这里，我们报告了一个新的选择标记，可以允许多次转染。我们已经利用了我们的发现，即恶性疟原虫红细胞期线粒体电子传递链（mtETC）的关键功能是作为二氢乳清酸脱氢酶（DHODH）的共底物的泛醌的再生，并且表达来自酵母的非泛醌依赖性DHODH（yDHODH）的转基因恶性疟原虫对所有mtETC抑制剂具有抗性。我们评估了使用yDHODH作为恶性疟原虫转染的阳性选择标记的可能性，包括其在基因破坏策略中的用途。我们构建了一个转染载体，设计用于基因破坏，称为pUF-1，含有yDHODH基因作为阳性选择标记，与先前描述的融合酵母胞嘧啶脱氨酶-尿嘧啶磷酸核糖转移酶基因作为阴性选择标记相结合。转染D10菌株，然后用阿托伐醌进行选择，得到阳性选择的含有质粒的寄生虫，表明yDHODH可用作选择性标记。然而，阿托伐醌不能用于恶性疟原虫Dd 2株的这种选择。另一方面，我们证明了yDHODH转基因寄生虫可以在两种菌株中通过疟原虫DHODH特异性的基于三唑并嘧啶的抑制剂来选择。因此，用DHODH抑制剂进行选择是上级的，因为它成功地选择了转基因Dd 2寄生虫，并且在较短的选择期后产生了转基因寄生虫。作为概念的证明，我们已经成功地破坏了II型液泡质子泵焦磷酸酶基因（PfVP 2）在恶性疟原虫的双交叉重组，表明该基因是不是血液阶段的寄生虫的生存所必需的。
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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影响因子：
64.8
作者：
Carlton, Jane M.;Adams, John H.;Silva, Joana C.;Bidwell, Shelby L.;Lorenzi, Hernan;Caler, Elisabet;Crabtree, Jonathan;Angiuoli, Samuel V.;Merino, Emilio F.;Amedeo, Paolo;Cheng, Qin;Coulson, Richard M. R.;Crabb, Brendan S.;del Portillo, Hernando A.;Essien, Kobby;Feldblyum, Tamara V.;Fernandez-Becerra, Carmen;Gilson, Paul R.;Gueye, Amy H.;Guo, Xiang;Kang'a, Simon;Kooij, Taco W. A.;Korsinczky, Michael;Meyer, Esmeralda V. -S.;Nene, Vish;Paulsen, Ian;White, Owen;Ralph, Stuart A.;Ren, Qinghu;Sargeant, Tobias J.;Salzberg, Steven L.;Stoeckert, Christian J.;Sullivan, Steven A.;Yamamoto, Marcio M.;Hoffman, Stephen L.;Wortman, Jennifer R.;Gardner, Malcolm J.;Galinski, Mary R.;Barnwell, John W.;Fraser-Liggett, Claire M.
通讯作者：
Fraser-Liggett, Claire M.
DOI：
10.1073/pnas.0610353104
发表时间：
2007-01-16
影响因子：
11.1
作者：
Hill, David A.;Pillai, Ajay D.;Desai, Sanjay A.
通讯作者：
Desai, Sanjay A.
影响因子：
1.5
作者：
McIntosh, MT;Drozdowicz, YM;Vaidya, AB
通讯作者：
Vaidya, AB
影响因子：
1.5
作者：
Maier, Alexander G.;Braks, Joanna A. M.;Cowman, Alan F.
通讯作者：
Cowman, Alan F.
影响因子：
64.8
作者：
Menard, R;Sultan, AA;Nussenzweig, V
通讯作者：
Nussenzweig, V