Expression of Green Fluorescent Protein in the Retina of Knock–In GFP–Rhodopsin Fusion Mice

Abstract

Abstract: : Purpose: Rhodopsin–green fluorescent protein (GFP) fusion (rho–gfp) mice have their native rhodopsin gene replaced by the human rhodopsin gene with its C terminus modified to encode an enhanced GFP fusion. GFP expression in these mice serves as a sensitive indicator of rod–cell structure and integrity. It may also serve as a marker for rod specific progenitor cells in the retina of these mice. We examined GFP expression in vivo and in vitro in the retina of rho–gfp mice. Methods: We explanted retina from postnatal day 1 (P1) rho–gfp mice and cultured the retina in defined media without EGF. The retinal explants were monitored daily for GFP expression by fluorescence microscopy. We also sacrificed rho–gfp mice on P0, P1, P3 and P5 to evaluate in vivo GFP expression, again by fluorescence microscopy. Finally, we isolated retinal progenitor cells from the retina of P1 rho–gfp mice using established methods and allowed them to expand for 10 days. We then sorted the cells by FACS based on GFP expression. Results: The P1 retinal explants began expressing GFP after 2 days in culture, and nearly all the cells in the photreceptor layer were GFP+ after 3 days in culture. In vivo expression of GFP in the retina of the rho–gfp mice was not significantly different. Expression of GFP was seen in the retina of enucleation specimens of P3 mice, and expression of GFP was seen in the majority of the photoreceptor cell layer starting at P5. On FACS analysis, approximately 30% of the retinal progenitor cells grown in culture were GFP+. Conclusions: Our results indicate that GFP expression in rho–gfp mice begins earlier than previously reported. These transgenic mice provide a valuable tool for study of rod development and may be also useful for the isolation of photoreceptor progenitor cells.

Keywords: retina: distal (photoreceptors, horizontal cells, bipolar cells) • retinal development