Glial Fibrillary Acidic Protein (GFAP) gene regulation in transfected Müller cell cultures

Purpose. The glial fibrillary acidic protein (GFAP) gene is induced in Müller cells in response to photoreceptor degeneration. The genetic elements responsible for GFAP gene activation are not known. Recent studies with transgenic mice carrying GFAP-lacZ fusion genes suggest that genetic elements that regulate GFAP expression in Müller cells are not present in immediate 5′ and 3′ flanking sequences of the GFAP gene. In the present study we have examined whether 5′, 3′ and intronic sequences of the mouse GFAP gene can drive expression of the lacZ reporter in Müller cell cultures. Methods. Lipofectin-mediated DNA transfection was used to examine gene expression in cell cultures. The Müller cell cultures used were obtained from animals with constant light damage, and expressed GFAP endogenously. C6 glioma cells were used as a positive control. Transfection with a control plasmid, pSVlac, in which β-gal is expressed under SV40 promoter was used to normalize all transfection data. Results. Three different GFAP-lacZ fusion genes were tested: C-339 carried the entire GFAP gene with 2 kb of 5′ and 1 kb of 3′ flanking sequence, and had lacZ inserted in the first exon; C-445 was similar to C-339 except that the first two ATGs were changed to TTGs; and C-259 had 2 kb of 5′ sequence fused directly to lacZ gene. In C6 cells transfected with the three different GFAP-lacZ constructs β-gal levels were elevated. C-339 showed a 40% stimulation whereas C-445 and C-259 gave 39% and 33% stimulation, respectively. Transfected Müller cell cultures showed a 6% change in β-gal activity. Conclusions. The immediate 5′-, 3′-flanking sequences and introns are not sufficient to direct GFAP gene expression in Müller cell cultures. These data are in agreement with in vivo studies and suggest that genetic elements that regulate GFAP expression in Müller cells and astrocytes are distinct.