Cellular dynamics visualized in live cells in vitro and in vivo by differential dual-color nuclear-cytoplasmic fluorescent-protein expression
Cellular dynamics visualized in live cells in vitro and in vivo by differential dual-color nuclear-cytoplasmic fluorescent-protein expression
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DOI：
10.1158/0008-5472.can-04-0643
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发表时间：
2004-06-15
期刊：
影响因子：
11.2
通讯作者：
Hoffman, RM
中科院分区：
文献类型：
--
作者：
Yamamoto, N;Jiang, P;Hoffman, RM
我们在这里报告的基因工程的双色荧光细胞的一种颜色在细胞核和其他在细胞质中，使实时的核质动力学可视化活细胞在体内以及在体外。为了获得双色细胞，在HT-1080人纤维肉瘤细胞的细胞质中表达红色荧光蛋白（RFP），在细胞核中表达与组蛋白H2 B连接的绿色荧光蛋白（GFP）。核GFP表达使核动力学可视化，而同时细胞质RFP表达使核质比可视化，以及同时细胞和核形状的变化。因此，可以在活的双色细胞中以真实的时间可视化总细胞动力学。亲本HT-1080和衍生的双色克隆具有相似的细胞增殖率，表明GFP和/或RFP的表达不影响细胞周期进程。单个活细胞的细胞周期位置很容易通过核质比和核形态可视化。实时诱导细胞凋亡观察核大小的变化和渐进的核碎裂。在小鼠耳中注射后，通过全身成像观察有丝分裂细胞。颈总动脉注射的双色细胞和可逆的皮瓣，使外部可视化的双色细胞在微血管中的小鼠大脑中的细胞体以及细胞核的极端伸长发生。尾静脉注射双色细胞后，在离体小鼠肺中观察到细胞周期不同位置的双色细胞。在肺中，经常观察到双色细胞并置其细胞核，这表明细胞间通讯的一种潜在的新形式。因此，双色细胞是一种有用的工具，用于可视化活细胞在体内和体外的动力学。能够特异性干扰这些过程的药物现在可以很容易地在体内真实的时间内进行筛选。
We report here the genetic engineering of dual-color fluorescent cells with one color in the nucleus and the other in the cytoplasm that enables real-time nuclear-cytoplasmic dynamics to be visualized in living cells in vivo as well as in vitro. To obtain the dual-color cells, red fluorescent protein (RFP) was expressed in the cytoplasm of HT-1080 human fibrosarcoma cells, and green fluorescent protein (GFP) linked to histone H2B was expressed in the nucleus. Nuclear GFP expression enabled visualization of nuclear dynamics, whereas simultaneous cytoplasmic RFP expression enabled visualization of nuclear cytoplasmic ratios as well as simultaneous cell and nuclear shape changes. Thus, total cellular dynamics can be visualized in the living dual-color cells in real time. The parental HT-1080 and the derived dual-color clones had similar cell proliferation rates, suggesting that expression of GFP and/or RFP does not affect cell cycle progression. The cell cycle position of individual living cells was readily visualized by the nuclear-cytoplasmic ratio and nuclear morphology. Real-time induction of apoptosis was observed by nuclear size changes and progressive nuclear fragmentation. Mitotic cells were visualized by whole-body imaging after injection in the mouse ear. Common carotid artery injection of dual-color cells and a reversible skin flap enabled the external visualization of the dual-color cells in microvessels in the mouse brain where extreme elongation of the cell body as well as the nucleus occurred. Dual-color cells in various positions of the cell cycle were visualized in excised mouse lungs after tail-vein injection of the dual-color cells. In the lung, the dual-color cells were observed frequently juxtaposing their nuclei, suggesting a potential novel form of cell-cell communication. The dual-color cells thus are a useful tool for visualizing living-cell dynamics in vivo as well as in vitro. Drugs that could specifically perturb these processes can now be readily screened in real time in vivo.