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J.E. Young, K.G. Gross, S.C. Khani; A Short Enhancer Segment of the Human Rhodopsin Kinase (Rk) Promoter Drives Robust Expression of Green Fluorescent Protein and Other Reporters in Transgenic Mouse Photoreceptors and Retinoblastoma Cell Lines . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3546.
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Purpose: Rhodopsin kinase (Rk) is a photoreceptor-specific enzyme that plays a key role in the light adaptation and recovery of photoreceptors. Spatial distribution of Rk appears to be dependent on the transcriptional activity of the promoter. The study below will aim to elucidate the minimal promoter region of the human Rk gene necessary to confer retina and photoreceptor-specific expression and to create model systems for further characterization of the Rk promoter elements and their interaction with the superseding regulatory factors. Methods: Deletion mutants of the 2.2-kb Rk promoter region were constructed, linked upstream of luciferase gene and lipofected into retinoblastoma cells. Luciferase activities were determined and compared. Transgenic mouse lines were produced using fragments of the human Rk promoter fused upstream of GFP and LacZ, which had shown activity in transfected cell culture experiments. Founder mice and subsequent generations were screened by PCR for the presence of the transgene. RT-PCR was used to evaluate transcription in ocular and various other tissues. Fluorescence of normal and GFP-positive mouse retinas were compared using an epifluorescent dissecting and confocal microscope. Immunostaining was further employed on fixed, cryo-sectioned whole globes to localize site of expression. LacZ expression in transgenic retinas was assessed using X-gal staining. Results: A short enhancer segment of approximately 0.11 kb conferred robust expression in WERI retinoblastoma cell lines. In transgenic mouse lines, RT-PCR analysis indicated transgene expression exclusively in the globe. Intense GFP fluorescence was noted in retina whole mounts of 0.11RKgfp mice. Moreover, immunostaining of whole globe sections revealed GFP localization in the photoreceptor layer of the transgenic mouse retina. Conclusions: The 0.11-kb 5’ flanking region of Rk contains the key elements sufficient for conferring accurate expression within WERI retinoblastoma and photoreceptors in transgenic mice.
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