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Maria M. De Lira, II, Leny J. Alvarez, Alejandro Martínez, Marisela Aguirre-Ramírez, Jorge A. Pérez-León; Expression And Regulatory Sites Of Melanopsin Gene In Rabbit Retina At Different Ages. Invest. Ophthalmol. Vis. Sci. 2012;53(14):6557.
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© ARVO (1962-2015); The Authors (2016-present)
Melanopsin (OPN4) is the pigment of photoreceptor ganglion cells of retina (ipRGC). The activity of these cells is not related to image formation but to photoentrainment of the oscillator within the suprachiasmatic nuclei. The ipRGCs have a dual function as photoreceptors and projection neurons; thus is interesting to know the regulation and expression of the OPN4 gene in animals that change from food-entrainment to photoentrainment during the early life, for example the rabbit. For this purpose is important to determinate a curve of the gene expression and an approximate age for protein activation. Likewise, would be important to know the sites of regulation of OPN4 gene using bioinformatics tools.
Rabbits ranging between 3 days to 4 months in age, caged with their mother and littermates were used in this project. They were kept in normal light-dark conditions. The retina was isolated from rabbits and the TRIZOL method was used for RNA extraction, followed by cDNA synthesis In order to perform conventional and quantitative PCR, specific primers for OPN4, Thy1 and GAPDH were designated and synthesised.
A fragment corresponding to exons 7/8 of the OPN4 gene was detected in cDNAs from all ages tested. This fragment was cloned in pGEMt vector and verified by sequencing and comparison to reported rabbit OPN4 sequence using clustal W 2.0. Hence the OPN4 gene is transcribed in rabbits, even before they open the eyes. We further performed qPCR assays for all genes. The expression of OPN4 keeps roughly the same level from P3 (3.59x104), then peaks at P12 (5.26x104), and lowers back until P32 (4.07x104). The GAPDH (9.01x103), gene has a constant expression level through all ages evaluated and the same was observed in Thy-1 expression level. The expression levels compared one to another gene showed that OPN4 is expressed at higher level relative to both GAPDH and Thy-1, at least ten-fold.
These data show a more abundant amount of OPN4 transcripts along the early development in rabbit. This could be because the cells that express OPN4 outnumber those expressing Thy-1, or due to a notably different ratio of expression of both genes by the same cells. In any case, the OPN4 transcripts are detected much more in advance to the ipRGCs role in photoentrainment. Attempts to identify a potential promoter and other gene regulatory sites of rabbit OPN4 gene are being made.
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