PDF(Pubmed)
Abstract：
Functional gene and protein characterizations in parasitic protists are often limited by their genetic tractability. Despite the development of CRISPR-Cas9-derived or inspired approaches for a handful of protist parasites, the overall genetic tractability of these organisms remains limited. The intestinal parasite Giardia lamblia is one such species, with the added challenge of a paucity of reliable selection markers. To address this limitation, we tested the feasibility of using Nourseothricin as an effective selection agent in Giardia. Here, we report that axenically-grown WB Giardia cells are sensitive to Nourseothricin and that engineering expression of the streptothricin acetyltransferase (SAT-1) gene from Streptomyces rochei in transgenic parasites confers resistance to this antibiotic. Furthermore, we determine that SAT-1-expressing parasites are cross-resistant neither to Neomycin nor Puromycin, which are widely used to select for transgenic parasites. Consequently, we show that Nourseothricin can be used in sequential combination with both Neomycin and Puromycin to select for dual transfection events. This work increases the number of reliable selection agents and markers for Giardia genetic manipulation, expanding the limited molecular toolbox for this species of global medical importance.
摘要：
寄生原生生物的功能基因和蛋白质特征通常受到其遗传可操作性的限制。尽管针对少数原生寄生虫开发了CRISPR-Cas9衍生或启发的方法，这些生物的总体遗传可操作性仍然有限。肠道寄生虫蓝氏贾第鞭毛虫就是这样一个物种，加上缺乏可靠的选择标记的额外挑战。为了解决这个限制，我们测试了在贾第虫中使用Nourseothricin作为有效选择剂的可行性。这里,我们报道,轴突生长的WB贾第鞭毛虫细胞对Nourserothricin敏感,并且在转基因寄生虫中工程化表达来自rochei链霉菌的链霉素乙酰转移酶(SAT-1)基因赋予了对该抗生素的抗性.此外，我们确定表达SAT-1的寄生虫对新霉素和嘌呤霉素都不具有交叉抗性，广泛用于选择转基因寄生虫。因此,我们表明,Nourseathricin可以与新霉素和嘌呤霉素连续联合使用,以选择双重转染事件.这项工作增加了可靠的选择剂和贾第虫遗传操作标记的数量，扩展这个具有全球医学重要性的物种的有限分子工具箱。
