Akutsu Yukie, Doi Motomichi, Furukawa Koji, Takagi Yuko
Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST).
Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST);
J Vis Exp. 2019 Jul 31(149). doi: 10.3791/59962.
Trypanosoma cruzi is a pathogenic protozoan parasite that causes Chagas' disease mainly in Latin America. In order to identify a novel drug target against T. cruzi, it is important to validate the essentiality of the target gene in the mammalian stage of the parasite, the amastigote. Amastigotes of T. cruzi replicate inside the host cell; thus, it is difficult to conduct a knockout experiment without going through other developmental stages. Recently, our group reported a growth condition in which the amastigote can replicate axenically for up to 10 days without losing its amastigote-like properties. By using this temporal axenic amastigote culture, we successfully introduced gRNAs directly into the Cas9-expressing amastigote to cause gene knockouts and analyzed their phenotypes exclusively in the amastigote stage. In this report, we describe a detailed protocol to produce in vitro derived extracellular amastigotes, and to utilize the axenic culture in a CRISPR/Cas9-mediated knockout experiment. The growth phenotype of knockout amastigotes can be evaluated either by cell counts of the axenic culture, or by replication of intracellular amastigote after host cell invasion. This method bypasses the parasite stage differentiation normally involved in producing a transgenic or a knockout amastigote. Utilization of the temporal axenic amastigote culture has the potential to expand the experimental freedom of stage-specific studies in T. cruzi.
克氏锥虫是一种致病性原生动物寄生虫，主要在拉丁美洲引起恰加斯病。为了确定针对克氏锥虫的新型药物靶点，验证靶点基因在寄生虫哺乳动物阶段即无鞭毛体中的必需性非常重要。克氏锥虫的无鞭毛体在宿主细胞内复制；因此，不经过其他发育阶段就很难进行基因敲除实验。最近，我们团队报道了一种生长条件，在此条件下无鞭毛体可以在无细胞环境中复制长达10天而不丧失其无鞭毛体样特性。通过使用这种临时无细胞无鞭毛体培养方法，我们成功地将引导RNA（gRNAs）直接导入表达Cas9的无鞭毛体中以实现基因敲除，并仅在无鞭毛体阶段分析其表型。在本报告中，我们描述了一个详细的方案，用于产生体外衍生的细胞外无鞭毛体，并在CRISPR/Cas9介导的基因敲除实验中利用无细胞培养。敲除无鞭毛体的生长表型可以通过无细胞培养的细胞计数或宿主细胞入侵后细胞内无鞭毛体的复制来评估。这种方法绕过了通常在产生转基因或基因敲除无鞭毛体时涉及的寄生虫阶段分化。利用临时无细胞无鞭毛体培养有可能扩大克氏锥虫阶段特异性研究的实验自由度。
