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A. Tsin, A. Muniz, A. Artis, A. Mukherjee; Expression of RPE65 and RGR in Cone-Dominated Chicken Retina and Primary Muller Cells. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2961.
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It has been shown that in the rod visual cycle the isomerases RGR and RPE65 play essential roles in the conversion of all-trans retinoids to the 11-cis form. The purpose of this study is to determine whether these enzymes are expressed in cone-dominated retina and Muller cells.
Muller cells were harvested and chicken retinas, and RPE were isolated from fresh chicken eyes. The RNA from each tissue was extracted and, from that, cDNA was synthesized using reverse transcription. Real time PCR was performed to amplify the cDNA obtained. Gel electrophoresis was performed to determine the PCR product size. The PCR product was also sequenced. We further analyzed the tissue and primary Muller cell samples by Western blot against RPE65.
The real time PCR plot showed that chicken RPE, retina, and Muller cells express both RGR and RPE65. Chicken RPE contains the greater level of expression of RGR and RPE65 and Muller cells have least. RPE cDNA required the least number of cycles to reach the threshold level for both RGR and RPE65. Also, RPE cDNA required the least number of fold-changes to reach threshold level followed by retina and Muller cells in both RGR and RPE65. The gel electrophoresis and use of the DNA ladder showed that RGR and RPE65 in all three tissues were the expected sizes, 221 and 281, respectively. We were able to confirm RPE65 protein in the chicken RPE, chicken retina and primary Muller cell cultures. Again, RPE has the highest level of RPE65 and the Muller cell having the lowest level of this protein.
The results of this study show that RGR and RPE65 are expressed in chicken retina and primary chicken Muller cells. The level of RPE65 protein reflects the levels of mRNA detected by RT-PCR.
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