Suarez Carlos E, McElwain Terry F
Program in Vector-Borne Diseases, Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA 99164-7040, USA.
Exp Parasitol. 2008 Apr;118(4):498-504. doi: 10.1016/j.exppara.2007.10.013. Epub 2007 Nov 5.
Transient transfection of intraerythrocytic Babesia bovis parasites has been previously reported. In this study, we describe the development and optimization of methods for transfection of purified B. bovis merozoites using either nucleofection (Amaxa) or conventional electroporation (Gene Pulser II, BioRad). Initially, the optimal buffer ("Plasmodium 88A6") and program (v-24) for nucleofection of free merozoites with a plasmid containing the luciferase gene as a reporter were determined. Using the same reporter plasmid, optimal voltage, capacitance and resistance for transfecting free merozoites by electroporation were defined to be 1.2 kV/25 microF/200 Omega. Using these optimal parameters, analysis of the time course of luciferase expression using either system to transfect free B. bovis merozoites showed high enzyme activity at 24h, with a rapid decline thereafter. Nucleofection was approximately five times more effective than electroporation when using a small quantity (2 microg) of DNA, while electroporation was twice as effective as nucleofection when a larger quantity of plasmid DNA (100 microg) was used. Parasite viability was significantly higher when using nucleofection when compared to electroporation regardless of the amount of DNA used. Comparison of luciferase expression after transfection of merozoites with circular, linearized, or double digested plasmid indicated that intact, circular plasmid was necessary for optimal luciferase expression. Overall, the results provide a basis for optimal transfection of purified B. bovis merozoites using either nucleofection or conventional electroporation. However, nucleofection is significantly more efficient when transfecting either circular or restriction digested DNA in the 2-10 microg range.
此前已有关于红细胞内牛巴贝斯虫寄生虫瞬时转染的报道。在本研究中，我们描述了使用核转染（Amaxa）或传统电穿孔（Gene Pulser II，Bio-Rad）对纯化的牛巴贝斯虫裂殖子进行转染方法的开发和优化。最初，确定了用于以含有荧光素酶基因作为报告基因的质粒对游离裂殖子进行核转染的最佳缓冲液（“疟原虫88A6”）和程序（v-24）。使用相同的报告质粒，将通过电穿孔转染游离裂殖子的最佳电压、电容和电阻定义为1.2 kV/25 μF/200 Ω。使用这些最佳参数，分析使用任一系统转染游离牛巴贝斯虫裂殖子后荧光素酶表达的时间进程，结果显示在24小时时酶活性较高，此后迅速下降。当使用少量（2 μg）DNA时，核转染的效率比电穿孔高约五倍，而当使用大量质粒DNA（100 μg）时，电穿孔的效率是核转染的两倍。无论使用的DNA量如何，与电穿孔相比，使用核转染时寄生虫活力显著更高。对用环状、线性化或双酶切质粒转染裂殖子后的荧光素酶表达进行比较表明，完整的环状质粒对于最佳荧光素酶表达是必需的。总体而言，这些结果为使用核转染或传统电穿孔对纯化的牛巴贝斯虫裂殖子进行最佳转染提供了基础。然而，当转染2-10 μg范围内的环状或限制性酶切DNA时，核转染的效率明显更高。
