Heterologously-expressed bacterial nitroreductase (NTR) enzymes sensitize eukaryotic cells to prodrugs such as metronidazole (MTZ), enabling selective cell ablation paradigms that have expanded studies of cell function and regeneration in vertebrate systems. However, first-generation NTRs require confoundingly toxic prodrug treatments (e.g. 10 mM MTZ) and some cell types have proven resistant. We used rational engineering and cross-species screening to develop a NTR variant, NTR 2.0, which exhibits ~100-fold improvement in MTZ-mediated cell-specific ablation efficacy. Toxicity tests in zebrafish showed no deleterious effects of prolonged MTZ treatments of ≤1 mM. NTR 2.0 therefore enables sustained cell loss paradigms and ablation of previously resistant cell types. These properties permit enhanced interrogations of cell function, extended challenges to the regenerative capacities of discrete stem cell niches, and enable modeling of chronic degenerative diseases. Accordingly, we have created a series of bipartite transgenic resources to facilitate dissemination of NTR 2.0 to the research community.

中文翻译：

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NTR 2.0：合理设计的前药转化酶，可显着增强靶向细胞消融的功效

异源表达的细菌硝基还原酶（NTR）酶使真核细胞对甲硝唑（MTZ）等前药敏感，从而实现了选择性的细胞消融范例，从而扩大了脊椎动物系统中细胞功能和再生的研究范围。但是，第一代NTR需要混杂的毒性前药治疗（例如10 mM MTZ），并且某些细胞类型已被证明具有抗药性。我们使用合理的工程设计和跨物种筛选来开发NTR变体NTR 2.0，该变体在MTZ介导的细胞特异性消融功效方面表现出约100倍的提高。斑马鱼的毒性试验表明，长期MTZ处理≤1mM不会产生有害影响。因此，NTR 2.0能够实现持续的细胞丢失范例，并消除先前耐药的细胞类型。这些特性可以增强对细胞功能的询问，离散的干细胞生态位的再生能力面临更大挑战，并能够对慢性退化性疾病进行建模。因此，我们创建了一系列两部分转基因资源，以促进NTR 2.0向研究界的传播。