离体电转染pCAGGS.EGFP对小鼠视网膜神经节细胞的标记 |
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引用本文: | 施圆圆,何士刚,李书华.离体电转染pCAGGS.EGFP对小鼠视网膜神经节细胞的标记[J].广州医学院学报,2013(4):10-12. |
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作者姓名: | 施圆圆 何士刚 李书华 |
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作者单位: | [1]广州医科大学生物技术实验中心.广东广州510182 [2]中科院生物物理研究所,北京100101 [3]广州医科大学病理教研室,广东广州510182 |
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基金项目: | 广州市教育局科技攻关项目:08A096 |
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摘 要: | 目的:通过离体电转染将GFP对胚胎期小鼠视网膜神经节细胞(RGC)进行标记来观察其形态.方法:剥离胚胎小鼠E14的视网膜连同晶状体,放入定制的电转杯中进行电转染,以8~16V电压将pCAGGS—EGFP质粒导入RGC,体外培养视网膜1~5d,观察绿色荧光蛋白GFP的表达情况。结果:以12V,50ms,950InS的间隔,5个脉冲转染效率最高,为81.25%:20~144h期间GFP依次由胞体向轴突、树突扩散分布。结论:在体外培养基中培养1~5d.pCAGGS—EGFP经过最适电压转染后能够在小鼠视网膜神经节细胞中持续扩散表达.
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关 键 词: | 视网膜神经节细胞 离体电转染 pCAGGS—EGFP质粒 体外培养 |
Electrotransfection using pCAGGS-EGFP plasmid for labeling retina neuroganglion in mice |
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Institution: | SHI Yuan-yuan , HE Shi-gang , L1 Shu-hua Center of Biotechnology, Guangzhou Medical University, Guangzhou 510182, China;2Biophysics Research Institute, Chinese Academy of Science, Beijing 100101, China;3Department of Pathology, Guangzhou Medical University, Guangzhou 5 1 0182, China |
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Abstract: | Objective :To label retina neuroganglion cells ( RGC ) with green fluorescent protein (GFP) in embryonic mice via electrotransfection, which entailed subsequent morphological assessments. Methods: The retina and lens in El4 mice were extracted in a specially prepared container for subsequent electrotransfection. The pCAGGS-EGFP plasmid was introduced into RGC by using a potential of 8-16 volts. This was followed by in vitro incubation of the retina for 1 to 5 days to determine the expression of GFP. Results : Electrotransfection with a potential of 12 volts and at 50ms and 950ms intervals for 5 pulsations was characterized by the highest efficiency (81.25 % ). GFP was distributed to the axons and dendrites between hour 20 and 144. Conelusion: pCAGGS- EGFP has a steady and effective expression in the mouse retinal ganglion cell ( RGC ) at embryonic stage for 5 days by electroporation in vitro at an optimal voltage. |
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Keywords: | retina neuroganglion electrotransfection in vitro pCAGGS-EGFP plasmid in vitro incubation |
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