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Y. Tang, N.M. Kumar; Microarray analysis for anti–cataract genes in the mouse lens . Invest. Ophthalmol. Vis. Sci. 2004;45(13):3983.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:To determine differences in transcript expression between the lenses of two mouse wild type strains (129 Sv and C57), as well as between Cx46 α3 KO and wild–type mice. The former comparison may lead to identification of potential candidate(s) genes that prevent (or promote) cataract formation, whereas the latter comparison may provide insights into the mechanism by which cataract formation occurs in the Cx46 α3 KO mice. Methods:Total RNA was isolated from lenses of 10 day mice. Affymetrix U74Av2 set was used for analysis of gene expression profiling of RNA isolated from lenses of wild type and α3 connexin knockout mice from both 129Sv and C57bl strains. Only the genes between experimental groups that have more than 1.5 fold change (increased or decreased) and where the p–value is less than 0.005 (increased) or larger than 0.995 (decreased) are reported. To confirm the validity of the transcript change determined by microarray analysis, 1–2 µg of total RNA was used for generation of cDNA by reverse transcriptase. This cDNA was used for real–time PCR using specific primers with detection of the PCR product by SYBR Green I dye. Results:Analysis of gene expression in the lenses from different strains and Cx46 α3KO mice indicated that there were more genes whose expression were changed in comparision of strains (129Sv vs C57) then in the comparision of the genes expressed in the Cx46 α3 knockout mice relative to the WT strains. Analysis of the microarray data indicated that the observed changes in transcript level between mice strains could be clustered into groups with specific functions, such as growth control. The transcript changes for a number of selected genes were confirmed by real–time PCR. Conclusions:The relatively small number of genes affected by disruption of the Cx46 α3 –connexin gene is consistent with a primary role for this connexin in maintaning lens homeostasis rather than in development. Comparison of the different strains indicated that genes that arrest cell growth and proliferation , such as GAS–5, TSC2, or affected by these processes were up regulated in the C57 strain. While the significance of these changes is not known, an hypothesis that explains the observed changes will be presented.
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