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M. Kuzmanovic, V.J. Dudley, V. Sarthy; GFP Expression in Retinal Müller Cells in GFAP-GFP Transgenic Mice . Invest. Ophthalmol. Vis. Sci. 2003;44(13):5178.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Glial fibrillary acidic protein (GFAP) is the best known marker for reactive gliosis in the retina. In order to study regulation of the GFAP gene in vivo, we have generated transgenic mice carrying a 2.5 kb, 5’ sequence of the mouse GFAP gene linked to the enhanced green fluorescent protein (EGFP) gene, and used the mice to examine GFP expression in adult and developing retina. Methods: A construct consisting of a 2.5 kb segment of the mouse GFAP promoter linked to the EGFP gene was designed. Transgenic mice were generated and screened by PCR to screen offspring that had successfully incorporated the transgene. Retinas from transgenic mice were isolated, fixed in paraformaldehyde and examined as whole mounts using fluorescence microscopy to determine GFP expression. In addition, 20µm frozen sections were cut and double-labeled with antibodies to several Muller cell markers to identify EGFP labeled cells. Results: In the adult retina, GFP labeling was seen in radially-oriented processes that scanned almost the entire thickness of the retina. Immunostaining with antibodies to CRALBP and glutamine synthetase showed that the GFP expressing cells were Müller cells. Furthermore, GFP-expressing Müller cells were observed in transgenic mice retinas in both the albino (CD1) and the pigmented (B6) background. These data suggest that the 2.5 Kb, 5’ sequence can direct reporter gene expression in Müller cells. Interestingly, there were few GFP-labeled astrocytes in the adult retina. However, in the developing retina, GFP labeled astrocytes were seen as early as P2 in the optic nerve head. As development progressed, these cells gradually moved towards the periphery of the retina and acquired their adult morphology. By P14 the entire surface of the retina was tiled by astrocytes. Conclusions: This study shows that the 2.5 kb, 5’ region of the mouse GFAP gene can be utilized to express GFP, and possibly other genes, in Müller cells. The GFAP-GFP mice will be useful for a variety of cell biological studies of Müller cells such their putative role as stem cells for retinal neurons. CR: None, Support: NIH EY-03523.
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