Plate-based transfection and culturing technique for genetic manipulation of Plasmodium falciparum.
Plate-based transfection and culturing technique for genetic manipulation of Plasmodium falciparum.
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DOI：
10.1186/1475-2875-11-22
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发表时间：
2012-01-18
期刊：
影响因子：
3
通讯作者：
DeRisi JL
中科院分区：
文献类型：
--
作者：
Caro F;Miller MG;DeRisi JL
疟原虫的基因操作仍然是一个低效、耗时且资源密集的过程。本文介绍了一套基于96孔板的转染和培养方法，提高了恶性疟原虫基因操作的效率。与标准方案相比，基于孔板的转染所需的DNA量少20倍，瞬时转染效率提高了约7倍，而稳定转染成功率在90%以上。此外，还展示了这套方案用于通过全细胞PCR筛选产生恶性疟原虫RH3假基因敲除的实用性。本文介绍的方法和工具将促进恶性疟原虫的基因组规模基因操作。
Genetic manipulation of malaria parasites remains an inefficient, time-consuming and resource-intensive process. Presented here is a set of methods for 96-well plate-based transfection and culture that improve the efficiency of genetic manipulation of Plasmodium falciparum. Compared to standard protocols plate-based transfection requires 20-fold less DNA, transient transfection efficiency achieved is approximately seven-fold higher, whilst stable transfection success rate is above 90%. Furthermore the utility of this set of protocols to generate a knockout of the PfRH3 pseudogene, screened by whole-cell PCR, is demonstrated. The methods and tools presented here will facilitate genome-scale genetic manipulation of P. falciparum.