Conditionally-immortalized astrocytic cell line expresses GAD and secretes GABA under tetracycline regulation.
We have engineered conditionally-immortalized mouse astrocytes to express beta-galactosidase or GAD(65) in a tetracycline-controlled fashion. The engineered cell lines, BASlinbetagal and BASlin65, divide at 33 degrees C but cease division at 39 degrees 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 BASlinbetagal cell line, we showed a dramatic regulation of beta-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.
Behrstock
S P
SP
Department of Physiological Science, University of California, Los Angeles, CA 90095-1761, USA.
Anantharam
V
V
Thompson
K W
KW
Schweitzer
E S
ES
Tobin
A J
AJ
D000818:Animals; D001253:Astrocytes / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D015153:Blotting, Western; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000096:biosynthesis / Q000235:genetics; D005455:Fluorescent Antibody Technique; D005968:Glutamate Decarboxylase / Q000096:biosynthesis* / Q000235:genetics; D051379:Mice; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology*; D013752:Tetracycline / Q000494:pharmacology*; D014162:Transfection; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics; D005680:gamma-Aminobutyric Acid / Q000378:metabolism*
D011500:Protein Synthesis Inhibitors; D005680:gamma-Aminobutyric Acid; D004247:DNA; D001616:beta-Galactosidase; D005968:Glutamate Decarboxylase; D013752:Tetracycline