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Conditionally‐immortalized astrocytic cell line expresses GAD and secretes GABA under tetracycline regulation
Author(s) -
Behrstock Soshana P.,
Anantharam Vellareddy,
Thompson Kerry W.,
Schweitzer Erik S.,
Tobin Allan J.
Publication year - 2000
Publication title -
journal of neuroscience research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.72
H-Index - 160
eISSN - 1097-4547
pISSN - 0360-4012
DOI - 10.1002/(sici)1097-4547(20000501)60:3<302::aid-jnr4>3.0.co;2-y
Subject(s) - immortalised cell line , cell culture , microbiology and biotechnology , cytoplasm , biology , tetracycline , transplantation , extracellular , glutamate decarboxylase , cell , biochemistry , enzyme , medicine , genetics , antibiotics
We have engineered conditionally‐immortalized mouse astrocytes to express β‐galactosidase or GAD 65 in a tetracycline‐controlled fashion. The engineered cell lines, BASlinβgal and BASlin65, divide at 33°C but cease division at 39°C. We carried out morphological and biochemical analyses to further understand GABA production and release, and to determine the suitability of these cells for transplantation. Using the BASlinβgal cell line, we showed a dramatic regulation of β‐galactosidase expression by tetracycline. The BASlin65 cell line showed functional GAD 65 enzymatic activity and GABA production, both of which were suppressed by growth in the presence of tetracycline. When cultured in the absence of tetracycline, BASlin65 cells have a total GABA content equal to or greater than other GABA‐ergic cell lines. Immunofluorescence microscopy revealed that GAD 65 had a distinct perinuclear localization and punctate staining pattern. GABA, on the other hand, showed diffuse staining throughout the cytoplasm. BASlin65 cells not only synthesize GABA, they also release it into the extracellular environment. Their ability to produce and release significant amounts of GABA in a tetracycline‐regulated manner makes BASlin65 cells a useful cellular model for the study of GABA production and release. Furthermore, their non‐tumorigenicity makes them excellent candidates for transplantation into specific regions of the brain to provide a localized and regulatable source of GABA to the local neuronal circuitry. J. Neurosci. Res. 3:302–310, 2000 © 2000 Wiley‐Liss, Inc.