GFAP GENE-EXPRESSION DURING DEVELOPMENT OF ASTROCYTE

Glial fibrillary acidic protein (GFAP) is expressed exclusively in ast rocytes in the central nervous system. In order to characterize indivi dual cultured cells in which the GFAP promoter is active and to identi fy the regulatory mechanisms of GFAP expression in these cells, we hav e developed a unique assay system for promoter activity using retrovir us vectors. Retrovirus containing the mouse GFAP promoter fused to the lacZ gene were used to infect mixed glial cultures. The infected cell s, in which the GFAP promoter was active, were visualized by X-Gal sta ining. From these experiments, we found that a 256 bp fragment 5' of t he transcription initiation site was sufficient to confer astrocyte-sp ecific expression of GFAP. The GFAP promoter became active about 3 day s before GFAP protein can be detected immunohistochemically, which ind icates that detection of GFAP promoter activity can be used to identif y astrocyte progenitors. We have also established immortalized astrocy te cell lines in which we detect GFAP promoter activity. Immorto mouse is a transgenic mouse generated by the introduction of thermolabile S V40 T Ag, tsA58. A mixed glial culture prepared from 2-day-old Immorto mouse brain was incubated at 32 degrees C, at which temperature most of the cells expressed T Ag. The culture was then infected with retrov irus containing GFAP promoter-lacZ, and the infected cells were select ed. Using the fluorescence-activated cell sorter with fluorescein di-b eta-D-galactopyranoside as a substrate (FDG-FACS), these cells were se parated into two groups: FDG(+), in which the GFAP promoter was active , and FDG(-), in which it was inactive. Mature astrocyte cell lines we re established from the FDG(+) cells by colony isolation. The FDG(-) c ells were cloned by colony isolation and cultured at 32 or 39 degrees C. At the latter temperature the expression of T Ag was suppressed and cell differentiation was induced in most cells. The cells which becam e positive for X-Gal staining only after switching to 39 degrees C wer e collected as immature astrocyte cell lines. These immortalized cell lines should be useful to investigate the molecular mechanisms of astr ocyte differentiation.