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Jeremy Whitson, Vincent M Monnier, Xingjun Fan; The Mouse Lens Glutathione Redox Transcriptome. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5599.
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© ARVO (1962-2015); The Authors (2016-present)
LEGSKO mice conditionally lack GCLC, and therefore the ability to synthesize GSH, in their lenses and develop cataract as a result of the low glutathione levels. In order to gain new insight into gene expression changes in response to low GSH levels and oxidative stress in cataractogenesis, a comparative transcriptome analysis of LEGSKO and WT lenses was carried out using RNA-Seq technology, and results were compared to microarray studies of human cataract as well as other mouse models of cataract.
Lenses were dissected, and epithelial and cortical fiber layers from two individual three month old LEGSKO and WT mice were separated. RNA was extracted using TRIZol reagent and cDNA was prepared using the Illumina system. Reads were mapped to mouse genome release mm10 using Tophat v2.0.11 after quality assessment using the FASTQC program. Differential gene expression analysis was done using Cuffdiff v2.2. RT-qPCR confirmation of this data is ongoing.
21014 transcripts were detected by the system in WT and/or LEGSKO epithelia while 17705 transcripts were detected in fiber cells. LEGSKO epithelia showed 417 significantly upregulated genes compared to wild-type and 92 downregulated genes. LEGSKO cortical fibers showed 277 significantly upregulated genes and 131 significantly downregulated genes. Functional categories of genes that were found to have modulated expression included: genes related to glutathione synthesis, metabolism, and usage, genes with a known linkage to cataract, hemoglobin and related genes, lens crystallins, genes encoding putative GSH transporters, antioxidant defense genes, genes involved in lens internal circulation, metal ion homeostasis genes, heat-shock protein and chaperone encoding genes, genes regulating apoptosis, genes involved in cell cycle, histone genes, protein synthesis genes, and genes involved in eye development.
There was a strong correlation between the RNA-Seq data and findings from microarray studies as well as many known cataract-related genes; confirming the relevancy of the LEGKSO model and giving new insights into the role of glutathione in cataractogenesis.
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