Conditionally-immortalized astrocytic cell line expresses GAD and secretesGABA under tetracycline regulation

We have engineered conditionally-immortalized mouse astrocytes to express P -galactosidase or GAD(65) in a tetracycline-controlled fashion. The enginee red cell lines, BASlin beta gal and BASlin65, divide at 33 degrees C but ce ase division at 39 degrees C. We carried out morphological and biochemical analyses to further understand GABA production and release, and to determin e the suitability of these cells for transplantation. Using the BASlin beta gal cell line, we showed a dramatic regulation of beta-galactosidase expre ssion by tetracycline. The BASlin65 cell line showed functional GAD(65) enz ymatic activity and GABA production, both of which were suppressed by growt h in the presence of tetracycline. When cultured in the absence of tetracyc line, BASlin65 cells have a total GABA content equal to or greater than oth er GABA-ergic cell lines. Immunofluorescence microscopy revealed that GAD(6 5) had a distinct perinuclear localization and punctate staining pattern. G ABA, on the other hand, showed diffuse staining throughout the cytoplasm. B ASlin65 cells not only synthesize GABA, they also release it into the extra cellular environment. Their ability to produce and release significant amou nts of GABA in a tetracycline-regulated manner makes BASlin65 cells a usefu l cellular model for the study of GABA production and release. Furthermore, their non-tumorigenicity makes them excellent candidates for transplantati on into specific regions of the brain to provide a localized and regulatabl e source of GABA to the local neuronal circuitry. (C) 2000 Wiley-Liss, Inc.