Kim Min Jung, Kang Kyung Ho, Kim Cheol-Hee, Choi Seok-Yong
Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA.
Biotechniques. 2008 Sep;45(3):331-4. doi: 10.2144/000112909.
Mitochondria maintain a web-shaped network in cells through a balance between fusion and fission. Under certain physiological and pathological conditions, this balance is breached, and as a result, change in mitochondrial morphology ensues. Real-time monitoring of such change is of significant importance for studying mitochondrial physiology and pathology, such as apoptosis, aging, and neurodegeneration. Numerous studies have been conducted in animal cell culture systems concerning mitochondrial morphology change. However, very little is known to date about the real-time changes in mitochondrial morphology at the organism level due to difficulties in observation and administration of mitochondria-disrupting drugs. Here we report the generation of transgenic zebrafish (Danio rerio) expressing mitochondrially targeted green fluorescent protein (GFP). The transparency of transgenic zebrafish embryos make it possible to monitor mitochondrial morphology in real time and in vivo. Since zebrafish inhabit fresh water, incubating zebrafish in drug-dissolved water sufficed to administer drugs to the zebrafish. We observed real-time and in vivo fragmentation of mitochondria in the transgenic embryos upon incubation in water with the following apoptosis-inducing drugs: valinomycin, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), and staurosporine. Thus, the transgenic zebrafish we generated could provide a platform for research on apoptosis and mitochondrial physiology and a screen for apoptosis-modulating drugs. It could also facilitate study of the pathogenesis of apoptosis-related diseases.
线粒体通过融合与分裂之间的平衡在细胞中维持网状网络。在某些生理和病理条件下，这种平衡被打破，结果线粒体形态发生改变。实时监测这种变化对于研究线粒体生理学和病理学，如细胞凋亡、衰老和神经退行性变具有重要意义。在动物细胞培养系统中已经进行了许多关于线粒体形态变化的研究。然而，由于观察和施用破坏线粒体的药物存在困难，迄今为止，关于生物体水平上线粒体形态的实时变化知之甚少。在这里，我们报告了表达线粒体靶向绿色荧光蛋白（GFP）的转基因斑马鱼（Danio rerio）的产生。转基因斑马鱼胚胎的透明性使得实时和在体内监测线粒体形态成为可能。由于斑马鱼生活在淡水中，将斑马鱼置于溶解有药物的水中就足以给斑马鱼施用药物。我们观察到，在用以下凋亡诱导药物处理的水中孵育时，转基因胚胎中的线粒体在实时和体内发生片段化：缬氨霉素、羰基氰4-（三氟甲氧基）苯腙（FCCP）和星形孢菌素。因此，我们产生的转基因斑马鱼可以为细胞凋亡和线粒体生理学研究提供一个平台，并用于筛选凋亡调节药物。它还可以促进对凋亡相关疾病发病机制的研究。
