Abstract
The purpose of this study is to develop a new approach for monitoring the function of implanted cells by a Renilla luciferase -glial cell line derived neurotrophic factor (RUC-GDNF) fusion protein in CSF. The RUC-GDNF protein is expected to report GDNF secreted from the cell by bioluminescence through luciferase activity.
Current ex vivo gene therapy using GDNF is limited by lack of a monitoring mechanism to determine the expression of GDNF once transformed cells or vector DNA are injected into animal models. Since the therapeutic concentration of GDNF secreted into cerebrospinal fluid (CSF) by implanted cells is too low for detection by the conventional methods such as ELISA and Western blotting, animals must be sacrificed to retrieve implanted cells in order to study their viability and functionality. Real-time monitoring of GDNF production by implanted cells in live animals was not possible.
To overcome the limits, a cell line named RG-1 was constructed by permanent transfection of glial cells with a plasmid DNA construct that contains a fusion gene encoding the RUC-GDNF fusion protein. The fusion protein is secreted by the RG-1 cell line and has both GDNF and RUC activity as determined by corresponding bioassays. The molecular weight of the fusion protein estimated by Western blotting was 70 to 75 kD, approximately the size of the sum of wild type GDNF and RUC. Concentrations of GDNF in the fusion protein as measured by ELISA correlate to light emitted by the same fusion protein as determined by RUC bioassay in the RG-1 culture medium. These findings indicate the suitability of RUC as the reporter in vitro. The cells were then stereotactically implanted to the anterior portion of Caudate nucleus of Sprague-Dawley rats. Cisternal CSF was collected before and up to 45 days after the cellular implantation.
We have obtained the following results: Bioluminescence in cisternal CSF was first detected at 6 hours post-cell implantation. The luciferase activity peaked at 24 to 48 hours, and then gradually declined after 10 days. Western blotting and ELISA tests failed to detect GDNF in CSF, suggesting that ELISA and Western blotting methods are insufficient in detecting GDNF at very low concentration.
This study demonstrates that the transformed glial cell line RG-1 offers the intrinsic “self-reporting” system for GDNF expression and transport in the rat brain. RUC secreted enables us to constantly monitor the function of the implanted cells in real time that promote the survival of dopaminergic cells in Parkinson’s Disease.
LLU Discipline
Pharmacology
Department
Pharmacology
School
Graduate School
First Advisor
Aladar A. Szalay
Second Advisor
Marvin Peters
Third Advisor
John Buchholz
Fourth Advisor
Robert Iacono
Fifth Advisor
Charles Slattery
Degree Name
Doctor of Philosophy (Medical Science)
Degree Level
Ph.D.
Year Degree Awarded
2000
Date (Title Page)
12-2000
Language
English
Library of Congress/MESH Subject Headings
Parkinson Disease -- therapy; Gene Therapy
Type
Dissertation
Page Count
x; 120
Digital Format
Digital Publisher
Loma Linda University Libraries
Copyright
Author
Usage Rights
This title appears here courtesy of the author, who has granted Loma Linda University a limited, non-exclusive right to make this publication available to the public. The author retains all other copyrights.
Recommended Citation
Liu, Hua, "Towards Gene Therapy of Parkinson’s Disease: Implantation of RUC-GDNF Secreting Mammalian Cell Lines into Rat Brain" (2000). Loma Linda University Electronic Theses, Dissertations & Projects. 1106.
https://scholarsrepository.llu.edu/etd/1106
Collection
Loma Linda University Electronic Theses and Dissertations
Collection Website
http://scholarsrepository.llu.edu/etd/
Repository
Loma Linda University. Del E. Webb Memorial Library. University Archives