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Delores Annette Davis, Steven J Pittler; RNA-seq analysis of retina and RPE in three different Cngb1 knockout mouse models. Invest. Ophthalmol. Vis. Sci. 2016;57(12):4798.
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© ARVO (1962-2015); The Authors (2016-present)
Retinitis pigmentosa (RP45) is caused by mutations of the cyclic nucleotide-gated cation channel β-subunit gene, which is vital for phototransduction. In rod photoreceptors, the Cngb1 locus encodes the β-subunit and two soluble proteins, GARP1 and GARP2. This study focuses on characterization of proteins/pathways involved in rod metabolism that involves the β-subunit and GARPs, and the identification of potential interactions.
Knockout mouse genotypes used in this study are: X1-/- (no β-subunit or GARPs); X26-/- (no β-subunit); and X12a-/- (no GARP2). RNA-NGS was performed on retina/RPE tissue extracted from PN 30 ± 1 d mice (n = 5) and analyzed on an Illumina HiSeq2000. Analysis was performed using Galaxy, PantherDB, and IGV 2.3 software. The cut-off for inclusion in further analysis was set at > 1.5 fold up- or down- regulation with a p-value < 0.05.
Comparison of all mutant datasets to WT identified 263, 201, and 165 genes that showed significant up or down differential transcription in X12a-/-, X26-/-, and X1-/-, respectively. Within these, X1-/- and X12a-/- share 44 genes, X1-/- and X26-/- share 31 genes, X12a-/- and X26-/- share 26 genes, and all three groups share 18 genes when collectively compared to WT. In X1-/- vs WT, the genes for previously reported down-regulated proteins Rho, Rom1, Gucy2d, and Pde6g are also down-regulated at the transcript level, however Prph2, Cnga1, and Abca4 levels were not different from WT. Gene ontology analysis of X12a-/- vs WT showed up-regulation of genes involved in cellular modification (15 genes), synaptic transmission (6), and DNA dependent transcription (23); with down-regulation of proteolytic enzymes (5), cell communication (12), and nervous (5) and muscular (5) system development.
Surprisingly, even though retinal degeneration is not apparent in X12a-/- mice, more differentially expressed genes were observed when compared to WT than the Cngb1 KOs that are progressively degenerating. This is consistent with the observed down-regulation of proteolytic genes and the lack of increase of apoptotic enzymes observed with retinal degeneration. The lack of correlation of transcript and protein levels observed for Prph2, Cnga1 and Abca4 in X1-/- indicates a post-transcriptional proteolytic mechanism that may account for the reduction in protein, without a corresponding change in transcript levels.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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