Krtková Jana, Paredez Alexander R
Department of Biology, University of Washington, Seattle, WA, 98195, USA.
Department of Experimental Plant Biology, Faculty of Science, Charles University in Prague, Viničná 5, 128 44, Prague 2, Czech Republic.
Methods Mol Biol. 2017;1565:123-140. doi: 10.1007/978-1-4939-6817-6_11.
Giardia lamblia, a major parasite, is an emerging model organism due to its compact genomic arrangement and composition. The most popular reverse genetic technique, RNAi, is ineffective in Giardia. In contrast, protein depletion by translation blocking morpholinos is suitable for most gene targets and provides up to 80% depletion of the target protein. The method is fast, reliable, and specific. After antisense morpholino oligomer delivery into Giardia trophozoites by electroporation, the cells can be used for many subsequent analyses 8-48 h after treatment. In this chapter, suitable gene tags, plasmids, and techniques necessary for proper morpholino targeting are described.
蓝氏贾第鞭毛虫是一种主要的寄生虫，由于其紧凑的基因组排列和组成，它正成为一种新兴的模式生物。最常用的反向遗传学技术RNA干扰在蓝氏贾第鞭毛虫中无效。相比之下，通过翻译阻断吗啉代寡核苷酸使蛋白质耗竭适用于大多数基因靶点，并能使靶蛋白耗竭高达80%。该方法快速、可靠且具有特异性。通过电穿孔将反义吗啉代寡聚物导入蓝氏贾第鞭毛虫滋养体后，处理后8 - 48小时的细胞可用于许多后续分析。在本章中，将描述合适的基因标签、质粒以及实现吗啉代寡核苷酸正确靶向所需的技术。
