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首页> 美国卫生研究院文献>PLoS Neglected Tropical Diseases >Utilization of proliferable extracellular amastigotes for transient gene expression drug sensitivity assay and CRISPR/Cas9-mediated gene knockout in Trypanosoma cruzi
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Utilization of proliferable extracellular amastigotes for transient gene expression drug sensitivity assay and CRISPR/Cas9-mediated gene knockout in Trypanosoma cruzi

机译：利用可增殖的胞外amastigotes用于瞬时基因表达药物敏感性测定和CRISPR / Cas9介导的克鲁氏锥虫基因敲除

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Trypanosoma cruzi has three distinct life cycle stages; epimastigote, trypomastigote, and amastigote. Amastigote is the replication stage in host mammalian cells, hence this stage of parasite has clinical significance in drug development research. Presence of extracellular amastigotes (EA) and their infection capability have been known for some decades. Here, we demonstrate that EA can be utilized as an axenic culture to aid in stage-specific study of T. cruzi. Amastigote-like property of axenic amastigote can be sustained in LIT medium at 37°C at least for 1 week, judging from their morphology, amastigote-specific UTR-regulated GFP expression, and stage-specific expression of selected endogenous genes. Inhibitory effect of benznidazole and nifurtimox on axenic amastigotes was comparable to that on intracellular amastigotes. Exogenous nucleic acids can be transfected into EA via conventional electroporation, and selective marker could be utilized for enrichment of transfectants. We also demonstrate that CRISPR/Cas9-mediated gene knockout can be performed in EA. Essentiality of the target gene can be evaluated by the growth capability of the knockout EA, either by continuation of axenic culturing or by host infection and following replication as intracellular amastigotes. By taking advantage of the accessibility and sturdiness of EA, we can potentially expand our experimental freedom in studying amastigote stage of T. cruzi.
机译：克氏锥虫具有三个不同的生命周期阶段。鞭毛体，锥鞭毛体和鞭毛体。鞭毛体是宿主哺乳动物细胞中的复制阶段，因此该寄生虫阶段在药物开发研究中具有临床意义。几十年来，人们已经知道细胞外变形虫（EA）的存在及其感染能力。在这里，我们证明了EA可以用作焦虑症培养物，以帮助克氏锥虫的特定阶段研究。从其形态，受mas虫特异性UTR调节的GFP表达和所选内源基因的阶段特异性表达来看，轴突性近日鞭毛虫的类似近日鞭毛虫的特性可以在37°C的LIT培养基中至少维持1周。苯并硝唑和硝呋替莫克斯对轴突性扁桃体的抑制作用与细胞内扁桃体的抑制作用相当。可以通过常规的电穿孔将外源核酸转染到EA中，并且可以使用选择性标记物富集转染子。我们还证明了可以在EA中进行CRISPR / Cas9介导的基因敲除。靶基因的必要性可以通过敲除EA的生长能力，通过持续的无菌培养或通过宿主感染，以及在复制后作为细胞内的变形虫来评估。通过利用EA的可访问性和坚固性，我们可以潜在地扩展在研究克鲁氏T假肢的阶段中的实验自由。

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