Comparison of the absolute and relative efficiencies of electroporation-based transfection protocols for Plasmodium falciparum
Comparison of the absolute and relative efficiencies of electroporation-based transfection protocols for Plasmodium falciparum
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DOI：
10.1186/1475-2875-11-210
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发表时间：
2012-06-21
期刊：
影响因子：
3
通讯作者：
Horrocks, Paul
中科院分区：
文献类型：
--
作者：
Hasenkamp, Sandra;Russell, Karen T.;Horrocks, Paul
背景：存在多种电穿孔方案可将外源 DNA 转染至恶性疟原虫中。然而，迄今为止，仅报道了对其相对效率的主观分析。方法：使用荧光素酶报告基因表达的时间过程来对三种电穿孔技术的绝对效率进行客观定量分析；环期感染红细胞的直接电穿孔、红细胞预加载以及结合这两种方法的新型“双击”方案。结果：红细胞预加载的平均效率为 9.59x10(-6)，比单独使用环期感染红细胞的“双击”和直接电穿孔的匹配实验分别效率高出约 5-180 倍。结论：此处提供的证据提供了首次对用于研究这种重要人类病原体的生物学和发病机制的关键分子工具的绝对和相对效率进行定量评估。
Background: Several electroporation protocols exist to transfect exogenous DNA into Plasmodium falciparum. To date, however, only a subjective analysis of their relative efficiencies has been reported.Methods: A time-course of luciferase reporter expression is used to provide an objective quantitative analysis of the absolute efficiency of three electroporation techniques; direct electroporation of ring stage infected erythrocytes, preloading of erythrocytes and a novel "double-tap" protocol that combines both approaches.Results: Preloading of erythrocytes shows a mean efficiency of 9.59x10(-6), some 5-180 fold more efficient than matched experiments utilizing the "double-tap" and direct electroporation of ring stage infected erythrocytes alone, respectively.Conclusion: Evidence presented here provides the first quantitative assessment of both the absolute and relative efficiencies of a key molecular tool used to study the biology and pathogenesis of this important human pathogen.