title: Detection of GDNF secretion in glial cell culture and from transformed cell implants in the brains of live animals.
abstract: 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.
authors.affiliation: Department of Biochemistry, Loma Linda University, School of Medicine, CA 92350, USA.
authors.forename: H
authors.initials: H
authors.lastname: Liu
authors.forename: R P
authors.initials: RP
authors.lastname: Iacono
authors.forename: A A
authors.initials: AA
authors.lastname: Szalay
mesh_terms: 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
chemical_list: 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