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Analysis of cell polarity formation by gene transfection in organs in situ

器官原位基因转染细胞极性形成分析

基本信息

批准号：
10470003
负责人：
TAKATA Kuniaki
金额：
$ 8.13万
依托单位：
Gunma University School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 2001
项目状态：
已结题

项目摘要

In vivo electroporation and particle gun methods were used to introduce exogenous genes into tissue cells in situ in living animals. Constructs of green fluorescent proteins were tested to optimize the conditions of gene transfection. Glucose transporters (GLUT1, GLUT3, GLUT4, GLUT5, and SGLT1) were introduced into cells of liver and pancreas of living rats. The expressed gene products were visualized by immunofluorescence staining of tissue sections. By optimizing the conditions of transfection, glucose transporters were successfully expressed in highly differentiated cells such as hepatocytes and pancreatic acinar cells forming tissues in situ. GLUT1 was localized to basolateral membrane domains in both hepatocytes and pancreatic acinar cells. GLUT3 and GLUT5 were restricted to apical membrane domain in pancreatic acinar cells. They were found in both the apical and basolateral domains in hepatocytes. These observations suggest differential targeting mechanism in hepatocytes and acinar cells : i.e., apical targeting is carried out by direct pathway in acinar cells, whereas it is mediated by the indirect transcytotic pathway in hepatocytes. GLUT4 was retained in the cytoplasmic compartments in both cell types. Immuno-electron microscopy revealed that GLUT4 resides in the membranes of zymogne secretary granules in acinar cells, showing that GLUT4 is sorted to the regulated secretion pathway in these cells. In addition, in vivo electroporation and particle gun methods are simple and versatile methods to study the localization of exogenous gene products in cells and tissues in situ.

采用体内电穿孔法和粒子枪法将外源基因原位导入活体动物的组织细胞中。构建绿色荧光蛋白，优化基因转染条件。将葡萄糖转运蛋白（GLUT 1、GLUT 3、GLUT 4、GLUT 5和SGLT 1）导入活体大鼠的肝脏和胰腺细胞。表达的基因产物通过组织切片的免疫荧光染色来可视化。通过优化转染条件，葡萄糖转运蛋白成功地在高分化细胞如肝细胞和胰腺腺泡细胞中原位表达。GLUT1定位于肝细胞和胰腺腺泡细胞的基底外侧膜域。GLUT3和GLUT5在胰腺腺泡细胞中局限于顶膜结构域。它们在肝细胞的顶侧和基底侧域都有发现。这些观察结果表明肝细胞和腺泡细胞中的差异靶向机制：即，顶端靶向在腺泡细胞中通过直接途径进行，而在肝细胞中通过间接转胞途径介导。GLUT 4保留在两种细胞类型的细胞质区室中。免疫电子显微镜显示，GLUT 4驻留在腺泡细胞中的酶原分泌颗粒的膜上，表明GLUT 4在这些细胞中被分选到受调节的分泌途径。此外，体内电穿孔和粒子枪方法是研究外源基因产物在细胞和组织中原位定位的简单而通用的方法。