Establishment of a transgenic zebrafish line for superficial skin ablation and functional validation of apoptosis modulators in vivo.
Establishment of a transgenic zebrafish line for superficial skin ablation and functional validation of apoptosis modulators in vivo.
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DOI：
10.1371/journal.pone.0020654
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发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Hsiao CD
中科院分区：
文献类型：
--
作者：
Chen CF;Chu CY;Chen TH;Lee SJ;Shen CN;Hsiao CD
斑马鱼皮肤由表皮层和基底层组成，它们构成了抵御病原体的第一线防御系统。斑马鱼表皮含有离子细胞和粘液细胞，有助于通过皮肤分泌酸/离子或粘液。先前的研究表明，鱼皮对外界刺激非常敏感。然而，对调节斑马鱼皮肤细胞凋亡的分子机制知之甚少。本研究旨在创建一个平台，进行条件性皮肤消融，并确定是否有可能通过在活体动物皮肤中过表达潜在的凋亡调节基因来减弱凋亡刺激。首先建立了转基因斑马鱼品系Tg（krt 4：NTR-hKikGR）cy 17（杀伤品系），其可以条件性地触发浅表皮肤细胞的凋亡。当杀伤细胞系与前体药物甲地那唑一起孵育时，通过检测大量TUNEL和活性半胱天冬酶3阳性信号判断，浅表皮肤显示出广泛的细胞凋亡。NTR-hKikGR+荧光信号的大幅减少伴随着表皮细胞凋亡。这表明NTR-hKikGR+信号荧光可用于评估体内凋亡事件。在去除甲地那唑后，皮肤完整性逐渐恢复，NTR-hKikGR+荧光信号逐渐恢复。相比之下，将杀伤细胞系与测试细胞系杂交或将杀伤细胞系与表达人组成型活性Akt 1、小鼠组成型活性Stat 3或HPV 16 E6元件的测试载体瞬时注射，显示出对细胞毒性甲氧咪挫的耐药表型，如通过NTR-hKikGR+荧光信号传导减少的损失所判断的。本研究中建立的杀伤细胞/测试线二元系统证明了硝基还原酶/甲氧咪啶唑系统可用于以实时方式有条件地进行皮肤消融，并提供了一种有价值的工具来可视化和量化体内感兴趣的靶基因的抗凋亡潜力。目前的工作确定了一个潜在的使用转基因斑马鱼作为一个高通量的平台，以验证潜在的凋亡调节剂在体内。
Zebrafish skin is composed of enveloping and basal layers which form a first-line defense system against pathogens. Zebrafish epidermis contains ionocytes and mucous cells that aid secretion of acid/ions or mucous through skin. Previous studies demonstrated that fish skin is extremely sensitive to external stimuli. However, little is known about the molecular mechanisms that modulate skin cell apoptosis in zebrafish. This study aimed to create a platform to conduct conditional skin ablation and determine if it is possible to attenuate apoptotic stimuli by overexpressing potential apoptosis modulating genes in the skin of live animals. A transgenic zebrafish line of Tg(krt4:NTR-hKikGR)cy17 (killer line), which can conditionally trigger apoptosis in superficial skin cells, was first established. When the killer line was incubated with the prodrug metrodinazole, the superficial skin displayed extensive apoptosis as judged by detection of massive TUNEL- and active caspase 3-positive signals. Great reductions in NTR-hKikGR+ fluorescent signals accompanied epidermal cell apoptosis. This indicated that NTR-hKikGR+ signal fluorescence can be utilized to evaluate apoptotic events in vivo. After removal of metrodinazole, the skin integrity progressively recovered and NTR-hKikGR+ fluorescent signals gradually restored. In contrast, either crossing the killer line with testing lines or transiently injecting the killer line with testing vectors that expressed human constitutive active Akt1, mouse constitutive active Stat3, or HPV16 E6 element displayed apoptosis-resistant phenotypes to cytotoxic metrodinazole as judged by the loss of reduction in NTR-hKikGR+ fluorescent signaling. The killer/testing line binary system established in the current study demonstrates a nitroreductase/metrodinazole system that can be utilized to conditionally perform skin ablation in a real-time manner, and provides a valuable tool to visualize and quantify the anti-apoptotic potential of interesting target genes in vivo. The current work identifies a potential use for transgenic zebrafish as a high-throughput platform to validate potential apoptosis modulators in vivo.
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影响因子：
4.8
作者：
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通讯作者：
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作者：
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影响因子：
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作者：
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通讯作者：
Hwang, Pung-Pung
影响因子：
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作者：
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通讯作者：
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