Production of Transgenic Medaka Fish Carrying Fluorescent Nuclei and Chromosomes
Production of Transgenic Medaka Fish Carrying Fluorescent Nuclei and Chromosomes
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DOI：
10.2108/zsj.26.9
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发表时间：
2009-01-01
影响因子：
0.9
通讯作者：
Yamashita, Masakane
中科院分区：
文献类型：
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作者：
Iwai, Toshiharu;Inoue, Shinya;Yamashita, Masakane
与斑马鱼一样，注意力集中在硬骨鱼青鳉作为生物科学各个领域中非哺乳类脊椎动物的实验动物代表。为了能够实时分析活青鳉细胞中的细胞核和染色体的动态，我们产生了在巨细胞病毒（CMV）启动子的控制下表达组蛋白H2 B和绿色荧光蛋白（GFP）之间的融合蛋白的转基因青鳉。由于转基因青鳉细胞的细胞核和染色体用GFP标记，因此可以在荧光显微镜下在整个有丝分裂细胞周期进程中即时监测它们的形态变化，而无需对样品进行任何固定和染色。然而，GFP标记的细胞核和染色体是不成功的早期胚胎发育，直到合子表达开始和减数分裂细胞周期的进程中，因为CMV启动子不工作，在这些阶段。此外，组蛋白H2 B-GFP融合蛋白以器官特异性方式表达;在包含肠和鳍的细胞中发生强烈和普遍的表达，而表达仅限于肝脏和大脑中的某些类型的细胞。这些发现表明，CMV驱动的组蛋白H2 B-GFP转基因的表达取决于转基因在基因组中的整合位点而被修饰。然而，通过使用转基因青鳉，在大多数有丝分裂细胞的细胞核和染色体的细胞学变化的简单和精确的监测将大大有助于更好地了解在脊椎动物细胞的核和染色体行为的控制机制。
As with zebrafish, attention has focused on the teleost medaka Oryzias latipes as an experimental animal representative of non-mammalian vertebrates in various fields of biological science. To enable real-time analyses of the dynamics of nuclei and chromosomes in living medaka cells, we produced a transgenic medaka expressing a fusion protein between histone H2B and green fluorescent protein (GFP) under the control of a cytomegalovirus (CMV) promoter. Since the nuclei and chromosomes of transgenic medaka cells are labeled with GFP, their morphological changes can be instantly monitored throughout the mitotic cell cycle progression under a fluorescent microscope without any fixation and staining of samples. However, GFP-labeling of nuclei and chromosomes is not successful during early embryonic development until zygotic expression begins and during the meiotic cell cycle progression, because the CMV promoter does not work in these stages. In addition, histone H2B-GFP fusion proteins are expressed in an organ-specific manner; strong and ubiquitous expression occurs in cells comprising the gut and fin, whereas the expression is restricted to certain types of cells in the liver and brain. These findings suggest that the CMV-driven expression of the histone H2B-GFP transgene is modified depending on the integration site of the transgene in the genome. Nevertheless, easy and precise monitoring of cytological changes in nuclei and chromosomes in the majority of mitotic cells by using the transgenic medaka will greatly contribute to a better understanding of control mechanisms of nuclear and chromosomal behaviors in vertebrate cells.