Enhanced Cell-Specific Ablation in Zebrafish Using a Triple Mutant of Escherichia Coli Nitroreductase
Enhanced Cell-Specific Ablation in Zebrafish Using a Triple Mutant of Escherichia Coli Nitroreductase
复制标题
DOI：
10.1089/zeb.2013.0937
复制
发表时间：
2014-04-01
期刊：
影响因子：
2
通讯作者：
Mumm, Jeff S.
中科院分区：
文献类型：
--
作者：
Mathias, Jonathan R.;Zhang, Zhanying;Mumm, Jeff S.
细菌硝基还原酶（NTR）的转基因表达促进化学诱导的靶向细胞消融。在斑马鱼中，NTR系统使细胞功能和细胞再生的研究成为可能。甲硝唑（MTZ）由于其细胞毒性衍生物的细胞特异性，已成为最常用的前药底物，用于诱导表达NTR的转基因斑马鱼的细胞损失。不幸的是，有效的细胞消融所需的MTZ治疗边界的毒性作用，因此，可能会招致不希望的非特异性效应。在这里，我们测试了NTR的三重突变体变体，先前在细菌测定中显示出改善的活性，是否可以通过使用减少的前药治疗方案促进斑马鱼中的细胞消融来解决这个问题。我们产生了几个互补的转基因斑马鱼线表达野生型或突变NTR（mutNTR）在特定的神经细胞类型，并测定前药诱导的细胞消融动力学使用共聚焦时间序列成像和板读数器为基础的定量荧光报告中表达的靶细胞类型。结果表明，在前药暴露时间显著缩短和/或前药浓度较低的情况下，可以在表达mutNTR的转基因系中实现细胞消融。这里表征的mutNTR变体可以避免由低前药转化效率引起的有问题的非特异性/毒性作用，从而增加这种选择性细胞消融方法的有效性和多功能性。
Transgenic expression of bacterial nitroreductase (NTR) facilitates chemically-inducible targeted cell ablation. In zebrafish, the NTR system enables studies of cell function and cellular regeneration. Metronidazole (MTZ) has become the most commonly used prodrug substrate for eliciting cell loss in NTR-expressing transgenic zebrafish due to the cell-specific nature of its cytotoxic derivatives. Unfortunately, MTZ treatments required for effective cell ablation border toxic effects, and, thus, likely incur undesirable nonspecific effects. Here, we tested whether a triple mutant variant of NTR, previously shown to display improved activity in bacterial assays, can solve this issue by promoting cell ablation in zebrafish using reduced prodrug treatment regimens. We generated several complementary transgenic zebrafish lines expressing either wild-type or mutant NTR (mutNTR) in specific neural cell types, and assayed prodrug-induced cell ablation kinetics using confocal time series imaging and plate reader-based quantification of fluorescent reporters expressed in targeted cell types. The results show that cell ablation can be achieved in mutNTR expressing transgenic lines with markedly shortened prodrug exposure times and/or at lower prodrug concentrations. The mutNTR variant characterized here can circumvent problematic nonspecific/toxic effects arising from low prodrug conversion efficiency, thus increasing the effectiveness and versatility of this selective cell ablation methodology.