High efficiency transfection of Plasmodium berghei facilitates novel selection procedures
High efficiency transfection of Plasmodium berghei facilitates novel selection procedures
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DOI：
10.1016/j.molbiopara.2005.09.007
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发表时间：
2006-01-01
影响因子：
1.5
通讯作者：
Waters, AP
中科院分区：
文献类型：
--
作者：
Janse, CJ;Franke-Fayard, B;Waters, AP
由于转染效率低（频率为10（-6）至10(-9)）和缺乏选择标记，转染技术在疟疾寄生虫生物学研究中的应用受到限制。在这里，一种新的转染方法，使用非病毒核因子(R)技术，描述了啮齿动物寄生虫伯氏疟原虫。获得的转染效率（episomal和targeted integration into genome）在10（-2）到10（-3）之间。如此高的转染效率大大减少了产生转染寄生虫所需的时间、实验动物数量和材料数量。此外，它允许开发不同的反向遗传学实验策略，我们展示了使用FACS直接选择稳定和不可逆转化的表达荧光蛋白（FP）的寄生虫。由于不需要使用药物选择性标记，该方法增加了（低）可用于伯格氏疟原虫转化的可选择标记的数量，原则上可以扩展到利用额外的FP。此外，facs选择的表达fp的寄生虫可以作为易于可视化的参考系，仍然可以用现有的药物选择标记进行遗传操作。提高转染效率和多用途啮齿动物模型的结合为进一步开发高通量基因组操作的新工具提供了基础。(c) 2005 Elsevier B.V.版权所有
The use of transfection in the study of the biology of malaria parasites has been limited due to poor transfection efficiencies (frequency of 10(-6) to 10(-9)) and a paucity of selection markers. Here, a new method of transfection, using non-viral Nucleofector (R) technology, is described for the rodent parasite Plasmodium berghei. The transfection efficiency obtained (episomal and targeted integration into the genome) is in the range of 10(-2) to 10(-3). Such high transfection efficiency strongly reduces the time, number of laboratory animals and amount of materials required to generate transfected parasites. Moreover, it allows different experimental strategies for reverse genetics to be developed and we demonstrate direct selection of stably and non-reversibly transformed, fluorescent protein (FP)-expressing parasites using FACS. Since there is no need to use a drug-selectable marker, this method increases the (low) number of selectable markers available for transformation of P. berghei and can in principle be extended to utilise additional FP. Furthermore the FACS-selected, FP-expressing parasites may serve as easily visualized reference lines that may still be genetically manipulated with the existing drug-selectable markers. The combination of enhanced transfection efficiency and a versatile rodent model provides a basis for the further development of novel tools for high throughput genome manipulation. (c) 2005 Elsevier B.V. All rights reserved.