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Vladlen Z Slepak, Alexey Pronin, Konstantin Levay, Qiang Wang, Dmitry Velmeshev, Mohammad Faghihi, Anurima Majumder, Kimberly Boyd, Nikolai Artemyev; Inhibition of transducin GTPase shifts global gene expression in the mouse retina. Invest. Ophthalmol. Vis. Sci. 2014;55(13):3031.
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© ARVO (1962-2015); The Authors (2016-present)
Molecular mechanisms linking rod photoreceptor cascade to photoreceptor cell death are not well understood. Here, we investigated changes in global gene expression that occur in mouse retina depending on illumination and in three genotypes: wild-type C57 BL6 mice, RGS9 knockout and transgenic mice expressing GTP-ase deficient transducin alpha subunit (GtQL).
Mice of the three genotypes were reared in complete darkness or normal (12 h light/12 h darkness) light cycle. After 8 weeks, total RNA was collected from the retinas and subjected to next generation sequencing using Illumina platform. The data was analyzed by CuffLinks and TopHat software to determine the number of reads and FPKM values. The expression of a selected number of genes was verified by quantitative RT-PCR. Retinal morphology was investigated by microscopy with H&E staining.
Retinal morphology remained normal across the genotypes showing the absence of major changes associated with retinal degeneration. We limited analysis to 5414 genes that expressed with FPKM of 10 and above. The levels of the majority of these genes varied less than 2-fold between dark-reared and control animals, in all three genotypes. In wild type animals 38 genes expressed > 2 fold higher in normal than in dark conditions; expression of 91 genes was down-regulated > 2-fold. In both RGS9 KO and GtQL animals the differences were significantly less: 20 and 22, and 19 and 34, respectively. Interestingly, there were genes that changed expression in all mutant animals compared to the wild type regardless of the light conditions. The largest fraction of genes down-regulated in mutant animals encodes proteins involved in protein synthesis, whereas the largest groups of genes up-regulated in mutant animals encode transcription factors and protein chaperones.
Gene expression pattern is different between mice reared in darkness vs. normal 12h light cycle. It appears that the prolonged GTP-bound state of transducin is sufficient to mimic the effect of light on global gene expression.
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