Puromycin-N-acetyltransferase as a selectable marker for use in Plasmodium falciparum
Puromycin-N-acetyltransferase as a selectable marker for use in Plasmodium falciparum
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DOI：
10.1016/s0166-6851(01)00344-9
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发表时间：
2001-10-01
影响因子：
1.5
通讯作者：
Crabb, BS
中科院分区：
文献类型：
--
作者：
de Koning-Ward, TF;Waters, AP;Crabb, BS
可用于疟疾转染的选择性标记数量有限，阻碍了恶性疟原虫基因组的广泛操作和随后对该生物体的彻底遗传分析。在本文中，我们表明，恶性疟原虫是高度敏感的药物嘌呤霉素，但嘌呤霉素-N-乙酰转移酶（PAC）基因的转基因表达链霉菌alboninger赋予这种药物的IC 50和IC 90值增加约3和7倍，分别在PAC表达寄生虫。尽管抗性水平相对较低，但用PAC选择性标记转染并直接在嘌呤霉素上选择的寄生虫群体在转染后以与用抗叶酸药物WR 99210独立选择的表达人二氢叶酸还原酶（hDHFR）的寄生虫相同的速率出现。转染的寄生虫通常以每个寄生虫2至6个拷贝的附加型维持PAC表达质粒。我们还通过在存在和不存在嘌呤霉素的情况下循环转染的寄生虫数周来证明，PAC选择标记可用于基因靶向。由于嘌呤霉素的作用模式不同于目前用于稳定转染恶性疟原虫的其它药物，PAC选择标记也应该具有与其它阳性选择标记结合使用的适用性，从而增加了对该生物体进行更复杂的功能研究的可能性。(C)2001 Elsevier Science B. V.保留所有权利。
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 IC50 and IC90 values increasing approximate to 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. (C) 2001 Elsevier Science B.V. All rights reserved.