1998 Fiscal Year Final Research Report Summary
Regulation of hypothalamic peptides by in vivo electroporation and its physiological effects
| Project/Area Number |
09671063
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (C)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 一般 |
|---|
| Research Field |
内分泌・代謝学
|
|---|
| Research Institution | Kagawa Medical University |
|---|
Principal Investigator |
SATO Makoto Kagawa Medical University, 1st Dept Int Med, Research Assosiate, 医学部, 助手 (10215840)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
TAKAHARA Jiro Kagawa Medical University, 1st Dept Int Med, Professor, 医学部, 教授 (00033085)
|
|---|
| Project Period (FY) |
1997 – 1998
|
| Keywords | electroporation / hypothalamus / oxytocin / vasopressin / beta-galactosidase / retrograde transport / rat / plasmid |
|---|
| Research Abstract |
To investigate physiological functions of hypothalamic peptides, it is important to control their expression at transcription levels. In this study, we attempted to establish in vivo electroporation to transfer an exogenous gene into hypothalamic nucleus in the rat. An expression vector, pCMVbeta, was used to examine the efficiency of gene transfer. The plasmid was injected into the lateral cerebral ventricle in rats and a bipolar electrode was inserted outside the hypothalamic arcuate nucleus (ARC). A series of electric pulses were delivered with a standard square wave electroporator. After the electroporation, brains of the rats were perfused with 4 % paraformaldehyde and cryostat sections were cut. The beta -galactosidase activity was examined using X-Gal histochemical stain. Staining tissues that express beta -galactosidase with X-Gal turned blue. Expression of beta -galactosidase was found in the choroid plexus, paraventricular nucleus (PVN), and supraoptic nucleus (SON). Although the electrode was located closely arround the hypothalamic ARC, the expression of beta -galactosidase was not observed in the ARC.Plasmid was also not incorporated into the ARC despite of the microinjection above the nucleus. The choroid plexus, PVN and SON showed positive expression of beta -galactosidase after injection of the plasmid into the carotid artery. These results suggest that the in vivo electroporation method might have a limited use for introducing exogenous genes in to the hypothalamic nucelus. The neurons that express beta -galactosidase in the PVN and SON appear to be oxytocin or vasopression, because these neurons are mostly abundant in both nuclei. The unexpected observation that the plasmid was incorporated into the PVN and SON raises the possibility that exogenous DNA may reach to these hypothalamic nuclei through their retrograde axonal transport. Further studies are required to establish a new method of in vivo gene transfer by the retrograde DNA transport.
|
