Detection of GDNF secretion in glial cell culture and from transformed cell implants in the brains of live animals.
Current ex vivo gene therapy for Parkinson's disease using glial cell line-derived neurotrophic factor (GDNF) is limited by the lack of a monitoring mechanism to determine the expression of GDNF once the cells or other vehicles are transferred into animal models. The purpose of this study was to test whether a Renilla luciferase (RUC)-GDNF fusion protein secreted by the genetically engineered glial cell line RG-1 could be measured photometrically in cerebrospinal fluid (CSF). RG-1 was constructed by permanent transformation with a plasmid DNA construct that contains a GDNF cDNA (gdnf) fused to a RUC cDNA (ruc). The fusion protein secreted by RG-1 was shown to retain both GDNF and RUC activity. The concentration of GDNF determined by enzyme-linked immunoadsorbent assay (ELISA) was correlated with the light emission detected by assaying for RUC bioluminescence in RG-1 culture medium, indicating that RUC can be used as a reporter for GDNF in vitro. The cells were then implanted into rat brain (n=20), and the cisternal CSF was analyzed. Bioluminescence was successfully detected in the CSF samples, and was quantified over a period of 25 days, while Western blotting and ELISA failed to detect GDNF in CSF, presumably because the concentration of the RUC-GDNF fusion was too low. This study demonstrates that the transformed glial cell line RG-1 offers a sensitive self-reporting assay for GDNF expression.
Liu
H
H
Department of Biochemistry, Loma Linda University, School of Medicine, CA 92350, USA.
Iacono
R P
RP
Szalay
A A
AA
D000818:Animals; D002460:Cell Line; D002555:Cerebrospinal Fluid / Q000378:metabolism; D017930:Genes, Reporter; D015316:Genetic Therapy / Q000379:methods; D051100:Glial Cell Line-Derived Neurotrophic Factor; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008297:Male; D051379:Mice; D017072:Neostriatum / Q000601:surgery; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D009457:Neuroglia / Q000378:metabolism* / Q000637:transplantation*; D010300:Parkinson Disease / Q000628:therapy; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014018:Tissue Distribution; D014170:Transformation, Genetic
C495040:Gdnf protein, mouse; C495041:Gdnf protein, rat; D051100:Glial Cell Line-Derived Neurotrophic Factor; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; D008156:Luciferases