The selectable marker human dihydrofolate reductase enables sequential genetic manipulation of the Plasmodium berghei genome
The selectable marker human dihydrofolate reductase enables sequential genetic manipulation of the Plasmodium berghei genome
复制标题
DOI：
10.1016/s0166-6851(99)00189-9
复制
发表时间：
2000-03-05
影响因子：
1.5
通讯作者：
Janse, CJ
中科院分区：
文献类型：
--
作者：
de Koning-Ward, TF;Fidock, DA;Janse, CJ
疟疾寄生虫的遗传转化受到可用的选择标记数量的限制。对于啮齿动物疟疾寄生虫。伯氏疟原虫（Plasmodium berghei），只有一个选择标记，利用来自伯氏疟原虫或刚地弓形虫的二氢叶酸还原酶-胸苷酸合酶基因赋予抗疟疾药物乙胺嘧啶抗性。在这里，我们报告使用人类二氢叶酸还原酶（hDHFR）基因作为一个新的选择性标记，赋予抗叶酸抑制剂WR 99210后乙胺嘧啶敏感和耐药菌株伯氏疟原虫。用含有hDHFR基因的环状构建体转染导致产生含有多个拷贝的附加型维持质粒的高度抗性寄生虫。与亲本寄生虫相比，这些寄生虫显示出对WR 99210的抗性增加约1000倍。我们还能够产生和选择仅携带单个拷贝的hDHFR靶向其基因组的转基因寄生虫，尽管这些寄生虫与亲本寄生虫相比仅显示出对WR 99210的抗性增加了五倍。重要的是，这是第一次在疟疾寄生虫中，hDHFR基因可以与现有的乙胺嘧啶选择标记结合使用。这通过将环子孢子（CS）基因重新引入含有伯氏疟原虫DHFR-TS选择标记的转基因CS敲除突变体寄生虫中来证明。hDHFR作为第二个选择性标记的开发将极大地扩展转化技术在疟原虫中的应用，使更广泛的遗传操作成为可能，从而促进对疟原虫生物学的更全面研究。(C)1000由Elsevier Science B. V.出版，保留所有权利。
Genetic transformation of malaria parasites has been limited by the number of selectable markers available. For the rodent malaria parasite. Plasmodium berghei, only a single selection marker has been at hand, utilising the dihydrofolate reductase-thymidylate synthase gene from either P. berghei or Toxoplasma gondii to confer resistance to the anti-malarial drug pyrimethamine. Here we report the use of the human dihydrofolate reductase (hDHFR) gene as a new selectable marker, which confers resistance to the antifolate inhibitor WR99210 upon both pyrimethamine sensitive and resistant isolates of P. berghei. Transfection with circular constructs containing the hDHFR gene resulted in the generation of highly resistant parasites containing multiple copies of episomally-maintained plasmids. These parasites showed around a 1000-fold increase in resistance to WR99210 compared to the parental parasites. We were also able to generate and select transgenic parasites harbouring only a single copy of hDHFR targeted into their genome, despite the fact that these parasites showed only a fivefold increase in resistance to WR99210 compared to the parental parasites. Importantly, and for the first time with malaria parasites, the hDHFR gene could be used in conjunction with the existing pyrimethamine selectable markers. This was demonstrated by reintroducing the circumsporozoite (CS) gene into transgenic CS-knockout mutant parasites that contained the P. berghei DHFR-TS selectable marker. The development of hDHFR as a second selectable marker will greatly expand the use of transformation technology in Plasmodium, enabling more extensive genetic manipulation and thus facilitating more comprehensive studies on the biology of the malaria parasite. (C) 1000 Published by Elsevier Science B.V. All rights reserved.