Purpose: To demonstrate that the crucial elements responsible for the spatial and temporal expression patterns of rhodopsin kinase (Rk) are contained within a narrow conserved segment immediately flanking the Rk transcription start sites.
Methods: Sequences upstream of the mouse Rk gene were compared to the human sequence to identify areas of conservation. Transgenic mice carrying a segment of the conserved human DNA sequence linked upstream of the green fluorescent protein (GFP) gene were examined by fluorescence microscopy and RT-PCR to localize GFP expression in retina and pineal gland. Rk and GFP temporal expression patterns were further compared by immunostaining and real-time RT-PCR in transgenic eyes during development.
Results: Comparison of the mouse and human 5' flanking sequence revealed only a small island of conserved sequence upstream of the respective Rk start sites. Uniform GFP expression was supported by a 0.2 kb fragment of the conserved human sequence in the transgenic mouse rods, cones, and pinealocytes. Developmental studies revealed an exponential rise in Rk and GFP transcripts in the first ten day postnatal period followed by a plateau later extending to adulthood. Rk and GFP proteins were first detected after postnatal day 10 and rose in parallel afterwards, overlapping in time with the maturation of photoreceptor outer segments and eye opening.
Conclusions: The conserved short enhancer/promoter immediately upstream of the Rk gene contains the key elements required for appropriate response to spatial and temporal cues during photoreceptor cell differentiation and fate determination. The above studies narrow the core sequences that govern gene expression in photoreceptors in vivo.