A rapid assay for glial cell line-derived neurotrophic factor and neurturin based on transfection of cells with tyrosine hydroxylase promoter-luciferase construct.
Glial cell line-derived neurotrophic factor (GDNF), a potent survival and trophic factor for various neuronal cells, has been measured by assaying its bioactivity based on neurite outgrowth or cell proliferation. We describe herein a sensitive and simple non-radioactive quantitative bioassay for GDNF family proteins based on their ability to induce tyrosine hydroxylase (TH) gene expression. Human neuroblastoma SK-N-MC cells were stably transfected with expression constructs of c-ret and with a luciferase reporter gene driven by 2 kb of the rat TH gene promoter region. In the presence of GDNF, luciferase activity increased with 20 h of incubation. A dose-dependent increase in luciferase activity was observed in the presence of GDNF between 1 and 300 ng/ml. This assay was also applicable for the quantification of the bioactivity of neurturin, another member of the GDNF family showing an even more sensitive profile of dose dependency than GDNF. Typical culture media were applicable in this assay. This method can be easily applied to numerous samples of conditioned medium in a short time.
Laboratory for Genes of Motor Systems, Bio-Mimetic Control Research Program, The Institute of Physical and Chemical Research (RIKEN), Moriyama, Nagoya 463-0003, Japan.
Mikiei
M
Tanaka
Hengyi
H
Xiao
Yoko
Y
Hirata
Kazutoshi
K
Kiuchi
D001681:Biological Assay / Q000379:methods*; D003470:Culture Media / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D017930:Genes, Reporter / Q000235:genetics; D051100:Glial Cell Line-Derived Neurotrophic Factor; D051097:Glial Cell Line-Derived Neurotrophic Factor Receptors; D006801:Humans; D008156:Luciferases / Q000235:genetics; D009414:Nerve Growth Factors / Q000032:analysis* / Q000494:pharmacology; D051101:Neurturin; D011401:Promoter Regions, Genetic / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000235:genetics; D051096:Proto-Oncogene Proteins c-ret; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics; D014162:Transfection / Q000379:methods*; D014407:Tumor Cells, Cultured; D014446:Tyrosine 3-Monooxygenase / Q000096:biosynthesis* / Q000235:genetics
D003470:Culture Media; C495039:GDNF protein, human; D051100:Glial Cell Line-Derived Neurotrophic Factor; D051097:Glial Cell Line-Derived Neurotrophic Factor Receptors; C495053:NRTN protein, human; D009414:Nerve Growth Factors; D051101:Neurturin; D011518:Proto-Oncogene Proteins; D008156:Luciferases; D014446:Tyrosine 3-Monooxygenase; D051096:Proto-Oncogene Proteins c-ret; D020794:Receptor Protein-Tyrosine Kinases