Abstract
Abstract: :
Purpose:In RPE, Cellular retinaldehyde binding protein (CRALBP) serves in the visual cycle as an 11–cis–retinol acceptor and as a substrate carrier. CRALBP is also expressed in Müller cells, ciliary epithelium, iris, cornea, pineal, brain and optic nerve. Toward deciphering the mechanisms controlling cell–specific expression of CRALBP, transgenic mice carrying mouse CRALBP promoter constructs have been generated and analyzed. Methods:Transgenic mice in strain B6CBA were generated in the Cleveland Clinic Transgenic Mouse Facility with EGFP reporter constructs containing either ∼1.4 Kb or ∼4.7 Kb of promoter from Rlbp1, the mouse gene encoding CRALBP. Southern blot and PCR were used for analysis of transgene expression. Founders were bred with mouse strains Balb/CbyJ or CD–1. Cryo sections of eye and brain tissues were processed for immunohistochemistry which was performed with antibodies to EGFP (BD Biosciences), GFAP (Sigma) and CRALBP. Nuclei were stained with TO–PRO–3 (Molecular Probes Inc). Results: Three independent founder mice were obtained for the 4.7Rlbp1–EGFP construct and two founders were obtained for the 1.4Rlbp1–EGFP construct. Breeding has yielded numerous progeny carrying the 4.7 Kb construct. Immunohistochemical analysis with anti–EGFP revealed GFP positive cells in the RPE and Müller cells of mice carrying the 4.7Rlbp1–EGFP construct. Concomitant labeling with CRALBP antibody was observed for many cells that showed expression of GFP or GFAP. Conclusions:The mouse CRALBP promoter region extending upstream from the transcription start site ∼4.7 Kb appears to direct expression to the RPE and Müller cells of the neural retina. Progress in on going histological analyses will be presented.
Keywords: gene/expression • retinoids/retinoid binding proteins • transgenics/knock–outs