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M Zheng, H Qian, J Nechtman, R Raten, SS Atherton; DNA Microarray Analysis of Mouse Contralateral Eye Following HSV-1 Anterior Chamber Inoculation . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3856.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To analyze the patterns of gene expression of 12488 genes in HSV-1 infected contralateral eye. Methods: Anterior chamber HSV-1 (KOS, 2 x 104 pfu/eye) inoculation was performed in the right eyes of BALB/c mice. On day 9 post inoculation, 10 contralateral eyes were enucleated and pooled and total RNA was isolated using Trizol. The same procedure was also applied for 10 contralateral eyes of mice whose ipsilateral eyes were inoculated with medium alone. cDNA was synthesized from 10 ug of total RNA using an oligo-dT primer containing a T7 RNA polymerase promoter. In vitro transcription was performed on ds-cDNA using the Enzo RNA transcript Labeling Kit. Biotin-labeled cRNA transcripts were purified, 20 ug was fragmented, and hybridized to a set of oligonucleotide arrays (Affimetrix). The chips were washed, stained with fluorescent anti-biotin and scanned. The levels of fluorescence for each gene were calculated using Affymetrix GeneChip Analysis software. Values representing expression of each gene were normalized. The normalized values were used to make a matrix-based decision concerning the expression level of an RNA molecule. Comparison analysis between test and control samples was made with Affimetrix software. The functional classifications were created by combining GeneSpring and Affimetrix software. Results: 5658 genes were upregulated at least 2 fold (≥2, ≤209) in 12488 genes studied in HSV-1 contralateral eyes compared with medium injected contralateral eyes. Among the 10 most upregulated genes, T cell specific protein and MHC II antigen A, MHC II k region locus 2 were upregulated 179, 164 and 162 fold, respectively. Three T cell receptor related genes were upregulated with the highest being 9.4 fold. MHC related genes were upregulated in 16 of 42 genes examined. Twenty-five cytokine and 16 chemokine genes were upregulated. Furthermore, 32 immunoglobulin genes and 9 complement component genes upregulated compared with the control eyes. Twenty-five macrophage related genes were also upregulated in experimental eyes. Conclusion: The above data provide additional support that T cells and immunomodulatory factors (cytokines, chemokines) are actively involved in HSV-1 retinitis. These results also suggest that B cells and/or macrophages may play a role in the pathogenesis of HSV-1 retinitis.
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