Efficient in utero gene transfer system to the developing mouse brain using electroporation:: Visualization of neuronal migration in the developing cortex
Efficient in utero gene transfer system to the developing mouse brain using electroporation:: Visualization of neuronal migration in the developing cortex
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DOI：
10.1016/s0306-4522(01)00016-1
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发表时间：
2001-01-01
期刊：
影响因子：
3.3
通讯作者：
Nakajima, K
中科院分区：
文献类型：
--
作者：
Tabata, H;Nakajima, K
我们报道了一种利用电穿孔的新型基因转移系统。我们使用该技术在胚胎12-17天不将胚胎从子宫取出的情况下，将含有增强绿色荧光蛋白的标记基因质粒导入小鼠大脑。这些胚胎在子宫内继续发育，超过80%的胚胎在出生时正常表达外源基因。由巨细胞病毒启动子驱动的增强绿色荧光蛋白在心室区、径向纤维和迁移的神经母细胞中强烈表达，但在成熟神经元中不表达，这表明巨细胞病毒启动子在细胞分化为成熟神经元后被沉默。由于目前还没有方便的方法来观察神经母细胞的迁移，特别是将它们与周围皮层板中的成熟神经元区分开来，该系统应该为分析神经元迁移提供一个很好的工具。在出生后外侧皮层，神经母细胞沿皮质板下斜向运行的“放射状”纤维几乎“切向”迁移，进入皮质板后转向边缘区并径向迁移。原始树突神经元仅在边缘区和皮质板之间的边界处被观察到，而在皮质板的中间等其他部位则未被观察到。这些结果表明，当神经母细胞遇到边缘区时，它们确实会停止迁移，并开始向成熟神经元分化，正如长期以来所建议的那样。相比之下，当使用延伸因子1 α启动子时，突出的荧光也允许整个成熟神经元的可视化。观察到标记的神经元向对侧皮层发送轴突，并在对侧皮层广泛分布。因此，该系统比病毒介导的基因转移更容易、更有效，可用于小鼠胚胎中神经细胞命运决定、迁移、定位和轴突寻径的功能获得分析。(c) 2001年。Elsevier Science Ltd.出版。版权所有。
We report a novel gene transfer system using electroporation. We used this technique to introduce a marker gene plasmid containing enhanced green fluorescent protein into mouse brains at embryonic day 12-17 without removing the embryos from the uterus. The embryos were allowed to continue to develop in utero, and more than 80% were born normally expressing the exogenous gene. Enhanced green fluorescent protein driven by the cytomegalovirus promoter was strongly expressed in the ventricular zone, radial fibers and migrating neuroblasts, but not in mature neurons, suggesting that the cytomegalovirus promoter is silenced after the cells differentiate into mature neurons. Since there is still no convenient way of visualizing the migrating neuroblasts, especially of distinguishing them from the surrounding mature neurons in the cortical plate, this system should provide a good tool for analysing neuronal migration. In the postnatal lateral cortex, neuroblasts migrated almost "tangentially" along the obliquely running "radial" fibers beneath the cortical plate, and after entering the cortical plate, turned towards the marginal zone and migrated radially. Neurons with primitive dendrites were observed only along the border between the marginal zone and the cortical plate, and never at other sites, such as in the middle of the cortical plate. These results imply that the neuroblasts do terminate migration and start differentiation to mature neurons when they encounter the marginal zone, as has long been suggested. By contrast, when elongation factor 1 alpha promoter was used, prominent fluorescence allowed visualization of the entire mature neurons as well. The labeled neurons were observed to send axons to the contralateral cortex where they arborized extensively.Thus, this system is much easier and more efficient than virus-mediated gene transfer, and is useful for gain-of-function analysis of neural cell fate determination, migration, positioning and axon path-finding in mouse embryos. (C) 2001 IBRO. Published by Elsevier Science Ltd. All rights reserved.