Nitroreductase-mediated Gonadal Dysgenesis for Infertility Control of Genetically Modified Zebrafish
Nitroreductase-mediated Gonadal Dysgenesis for Infertility Control of Genetically Modified Zebrafish
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DOI：
10.1007/s10126-009-9244-8
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发表时间：
2010-10-01
影响因子：
3
通讯作者：
Wu, Jen-Leih
中科院分区：
文献类型：
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作者：
Hu, Shao-Yang;Lin, Pei-Yu;Wu, Jen-Leih
转基因鱼在水产养殖中具有快速生长、抗病、耐寒等优良特性。然而，由于全球对食品安全和生态影响评估不完整的担忧，商业上可获得的转基因鱼受到限制。对生态影响的关注包括转基因鱼类的基因流动和逃逸，这可能导致野生天然鱼类种群灭绝。不孕不育控制是克服这一障碍的核心技术。虽然多倍体技术、GnRH特异性反义RNA和针对GnRH基因表达的RNAi已被用于导致鱼类不育，但这些方法并不是100%可靠的，而且不能遗传。本研究以斑马鱼为模型，建立转基因鱼不育控制的诱导型平台。将将甲硝唑底物转化为细胞毒素的硝基还原酶与绿色荧光蛋白融合，并在透明带启动子的作用下由卵母细胞特异性表达。在孵化后28~42d连续浸泡甲硝唑后，卵母细胞特异性的EGFP表达被消除，解剖分析发现性腺萎缩。这些发现表明，卵母细胞特异性硝基还原酶介导的甲硝唑催化阻止了卵子的发生，并导致未发育的卵母细胞。TUNEL法检测卵母细胞通过凋亡引起的死亡。我们发现，甲硝唑诱导的性腺发育不全导致卵巢杀伤基因bok的激活，该基因是bcl2家族的促凋亡基因成员，导致不育。这些结果表明，卵母细胞特异性硝基还原酶介导的甲硝唑催化可以导致斑马鱼可靠的不育，并有可能作为其他水产养殖鱼类的模型。
Genetically modified (GM) fish with desirable features such as rapid growth, disease resistance, and cold tolerance, among other traits, have been established in aquaculture. However, commercially available GM fish are restricted because of global concerns over the incomplete assessments of food safety and ecological impact. The ecological impact concerns include gene flow and escape of the GM fish, which may cause extinction of wild natural fish stocks. Infertility control is a core technology for overcoming this obstacle. Although polyploidy technology, GnRH-specific antisense RNA, and RNAi against GnRH gene expression have been used to cause infertility in fish, these approaches are not 100% reliable and are not heritable. In the present study, zebrafish was used as a model to establish an inducible platform of infertility control in GM fish. Nitroreductase, which converts metronidazole substrate into cytotoxin, was fused with EGFP and expressed specifically by oocytes in the Tg(ZP:NTR-EGFP) by a zona pellucida promoter. Through consecutive immersion of metronidazole from 28 to 42 days posthatching, oocyte-specific EGFP expression was eliminated, and atrophy of the gonads was detected by anatomical analysis. These findings reveal that oocyte-specific nitroreductase-mediated catalysis of metronidazole blocks oogenesis and leads to an undeveloped oocyte. Furthermore, oocyte cell death via apoptosis was detected by a TUNEL assay. We found that the gonadal dysgenesis induced by metronidazole resulted in activation of the ovarian killer gene bok, which is a proapoptotic gene member of the Bcl-2 family and led to infertility. These results show that oocyte-specific nitroreductase-mediated catalysis of metronidazole can cause reliable infertility in zebrafish and could potentially be used as a model for other aquaculture fish species.