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In vivo transfection of foreign genes into the neurons of rat brain as the neurografting method

将外源基因体内转染至大鼠脑神经元作为神经移植方法

基本信息

批准号：
03670684
负责人：
MIZUKAWA Kiminao
金额：
$ 0.19万
依托单位：
Okayama University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1992
项目状态：
已结题

项目摘要

First of all, I investigated the several factors controlling the efficiency of DNA transfection. I tried to use plasmid DNA as a vector for the introduction of genes into mouse muscles cells using cationic lipid-mediated DNA transfection(lipofection).By this improvement of this lipofectin-treated DNA, plasmid DNA were easily First of all, I investigated the several factors controlling the efficiency of DNA transfection. I tried to use plasmid DNA as a vector for the introduction of genes into mouse muscles cells using cationic lipid-mediated DNA transfection(lipofection).By this improvement of this lipofectin-treated DNA, plasmid DNA were easily injected into the mouse muscles through a microsyringe, and could be incorporated and expressed by muscle cells in the vicinity of injection point Morphologically I used two kinds of plasmid DNAs, L7RH-beta-gal and pRSVL-beta-gal, in order to detect the expression of transfected DNAs in mouse muscles cells. The L7RH-beta-gal plasmid DNA contain … More s the unclear location signal from the SV40 T-antigen gene and E.coli-beta-galactosidase gene both of which are fused to the SV40 virus enhancer-promoter. Since the produced E.coli beta-galactosidase proteins can be transported into nuclei of cells when the muscle cells were transfected with the L7RH-plasmid DNA, the nuclei should be densely stained with X-gal in a round shape. On the contrary, using the pRSVL-beta-gal, several number of the X-gal-stained mouse muscles fiber bundles were observed as blue colored muscles by light microscopy. By electron microscopic investigation, the large number of fine electron dense materials were occupied in the sarcolemma of the X-gal stained muscles.Although we have not identified any factors controlling the efficiency of lipofectin to mouse muscles cells with plasmid DNAs, improvements of this technique would provide us with a powerful tool for studying gene action in particular muscle cells in relation to morphological and functional aspects.Next stage, I will intend to apply this procedure to the neurografting. Less

首先，研究了影响DNA转染率的几个因素。本研究尝试以质粒DNA为载体，通过阳离子脂质体介导的DNA转染法(Lipofect)将基因导入小鼠肌肉细胞。通过对这种经脂质体处理的DNA的改进，首先考察了控制DNA转染率的几个因素。本研究通过阳离子脂质体介导的DNA转染法将基因导入到小鼠肌肉细胞中，通过对脂质体处理DNA的改进，可以方便地通过微量注射器将DNA注入小鼠肌肉，并在注射点附近的肌肉细胞形态上整合和表达。L7RH-β-GAL含有…S对SV40T抗原基因和大肠杆菌-β-半乳糖苷酶基因的定位不明信号进行了分析，这两个基因都与SV40病毒的增强子-启动子融合。由于将L7RH-质粒DNA导入肌细胞后，所产生的大肠杆菌β-半乳糖苷酶蛋白可转运到细胞核内，因此细胞核应以X-Gal浓染为圆形。反之，利用pRSVL-β-Gal，在光镜下可观察到一些X-Gal染色的小鼠肌肉纤维束呈蓝色。通过电子显微镜观察，X-Gal染色的肌肉肌膜内有大量细小的电子致密物质。虽然我们还没有发现任何控制脂质体DNA对小鼠肌肉细胞效率的因素，但这项技术的改进将为我们从形态和功能方面研究基因特别是肌肉细胞的作用提供了有力的工具。下一步，我打算将这一方法应用于神经移植。较少