2001 Fiscal Year Final Research Report Summary
Analysis of cell polarity formation by gene transfection in organs in situ
| Project/Area Number |
10470003
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
General anatomy (including Histology/Embryology)
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| Research Institution | Gunma University School of Medicine |
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Principal Investigator |
TAKATA Kuniaki School of Medicine, Gunma University, Professor, 医学部, 教授 (20129290)
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| Co-Investigator(Kenkyū-buntansha) |
SUZUKI Takeshi School of Medicine, Gunma University, Research Associate, 医学部, 助手 (00261868)
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| Project Period (FY) |
1998 – 2001
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| Keywords | liver / pancreas / electroporation / glucose transporters / immunohistochemistry / particle gun / immuno-electron microscopy |
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| Research Abstract |
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.
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