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D. Sharon, D. Bandah, E. Banin, G. Ben–Shlomo, R. Ofri; Spatial and Temporal Differential Gene Expression Analysis of PAX6 Alternatively–Spliced Transcripts in the Pigeon Retina . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5413.
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The molecular events leading to the development of different retinal regions, and particularly the macula, are poorly understood. We have chosen the pigeon retina, which includes both a central macula and a large nasal macular–like region (the red area), as a model for studying spatial and temporal retinal gene expression. Our purpose is to perform a detailed analysis of PAX6 alternatively–spliced isoforms in different regions of the pigeon retina.
Different retinal regions were dissected from pigeons with the pecten as an indicator of topography. Total RNA was extracted and used to produce cDNA by reverse transcriptase. Based on the published sequence of retinal genes in chicken, as well as in other species, we designed degenerated primers which were used to amplify the pigeon PAX6. PCR products were sequenced using an ABI 3700 DNA sequencer. The TA–cloning procedure was applied followed by sequencing of insert DNA. Quantitative real time RT–PCR analysis was performed on ABI–PRISM 7000 with cDNA samples from different retinal and brain regions calibrated for GAPDH expression level.
We have determined the sequence of the canonical pigeon PAX6 open reading frame, which is highly similar to human and chicken PAX6. A screen for alternatively–spliced isoforms revealed so far 3 alternatively–spliced sites producing 8 different transcripts, and potentially 8 functional PAX6 proteins. This includes the PAX(5a) isoform with an addition of an exon with 42 nucleotides, a skipping of 201 nucleotides in exon 6, and skipping of 18 nucleotides in exon 11. We have designed primers specific for each PAX6 isoform and verified that all 8 isoforms exist in the pigeon retina and brain. Quantitative real–time RT–PCR analysis revealed that all isoforms have a higher expression level in the retina versus the brain, and that 4 isoforms show significant differential expression in either adult versus young retina or macular red area versus peripheral retina.
Our analysis revealed two important findings regarding the involvement of PAX6 in retinal development:1. At least 8 different PAX6 isoforsm exist in the pigeon retina, and 2. At least 4 of which show differential expression levels in different retinal regions. The biological significance most of these splice variants is currently unknown, but taking into account the important role of PAX6 in ocular and brain development, we predict that some of these isoforms are involved in retinal , and possibly macular, development.
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