Pacheco-Lugo Lisandro, Díaz-Olmos Yirys, Sáenz-García José, Probst Christian Macagnan, DaRocha Wanderson Duarte
Laboratório de Genômica Funcional de Parasitos, Universidade Federal do Paraná, Brazil; Universidad Simón Bolívar, Barranquilla, Colombia.
Laboratório de Genômica Funcional, Instituto Carlos Chagas, FIOCRUZ Paraná, Brazil.
Parasitol Int. 2017 Jun;66(3):236-239. doi: 10.1016/j.parint.2017.01.019. Epub 2017 Jan 27.
New opportunities have raised to study the gene function approaches of Trypanosoma cruzi after its genome sequencing in 2005. Functional genomic approaches in Trypanosoma cruzi are challenging due to the reduced tools available for genetic manipulation, as well as to the reduced efficiency of the transient transfection conducted through conventional methods. The Amaxa nucleofector device was systematically tested in the present study in order to improve the electroporation conditions in the epimastigote forms of T. cruzi. The transfection efficiency was quantified using the green fluorescent protein (GFP) as reporter gene followed by cell survival assessment. The herein used nucleofection parameters have increased the survival rates (>90%) and the transfection efficiency by approximately 35%. The small amount of epimastigotes and DNA required for the nucleofection can turn the method adopted here into an attractive tool for high throughput screening (HTS) applications, and for gene editing in parasites where genetic manipulation tools remain relatively scarce.
2005年克氏锥虫基因组测序完成后，出现了研究其基因功能方法的新机遇。由于可用于基因操作的工具有限，以及通过传统方法进行瞬时转染的效率较低，克氏锥虫的功能基因组学方法具有挑战性。在本研究中，对Amaxa核转染仪进行了系统测试，以改善克氏锥虫前鞭毛体形式的电穿孔条件。使用绿色荧光蛋白（GFP）作为报告基因对转染效率进行定量，随后评估细胞存活率。本文使用的核转染参数提高了存活率（>90%），转染效率提高了约35%。核转染所需的前鞭毛体和DNA量较少，这使得本文采用的方法成为高通量筛选（HTS）应用以及寄生虫基因编辑的有吸引力的工具，因为寄生虫的基因操作工具仍然相对匮乏。
