Transcription profiling of mouse to identify strain-specific variation and the onset of gap junction mutation-induced cataracts

Disruption of the mouse gene encoding the gap junction subunit alpha3 connexin 46 (Cx46) results in the formation of lens cataracts. The timing of the onset of this lens opacity is affected by the genetic background, i.e. the mouse strain. To elucidate the mechanism by which cataracts form in the 129Sv/Jae strain earlier than in the C57BL/6J strain, global gene expression was quantitated in the lenses of these strains. Lens cDNAs were analyzed by hybridization to DNA microarrays and with real time-PCR. Theories are proposed based on the observed higher level of expression of the stress-response genes in the C57BL/6J strain and variations in the expression levels of genes involved in protein synthesis, metabolism, catabolism and cell proliferation. How these variations in gene expression might affect the response of lens fiber cells to the increased calcium, caused by lack of alpha3Cx46, is considered. The possibility that the proteins coded by the strain-variable genes might influence the cataract-associated proteolysis of gamma-crystallin is also addressed. Experiment Overall Design: To determine differences in transcript expression between the lenses of two mouse wild type strains (129 SvJae and C57BL/6J), as well as between alpha3Cx46 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 alpha3Cx46 KO mice. Total 8 samples were used ( two separate samples for each of the following 4 types of mice: 129SvJae wild type, 129SvJae alpha3Cx46 KO, C57BL/6J wild type and C57BL/6J alpha3Cx46 KO)敲除编码间隙连接亚基α3连接蛋白46（Cx46）的小鼠基因，可导致晶状体白内障的形成。此类晶状体混浊的发病时间受遗传背景，即小鼠品系，的影响。为阐明129Sv/Jae品系白内障发病早于C57BL/6J品系的分子机制，研究人员对这两个品系的晶状体开展了全局基因表达定量分析。通过DNA微阵列杂交与实时定量PCR（real time-PCR）对晶状体cDNA进行了检测分析。研究人员基于C57BL/6J品系中应激反应基因表达水平更高，以及蛋白质合成、代谢、分解代谢与细胞增殖相关基因的表达水平差异，提出了相关理论假说。研究同时探讨了此类基因表达差异可能如何影响晶状体纤维细胞对α3Cx46缺失所引发的钙水平升高的应答反应。此外，本研究还讨论了品系差异表达基因所编码的蛋白质可能影响与白内障相关的γ-晶体蛋白蛋白水解过程的可能性。实验整体设计：本实验旨在探究两种野生型小鼠品系（129SvJae与C57BL/6J）的晶状体之间，以及α3Cx46基因敲除（KO）小鼠与野生型小鼠的晶状体之间的转录本表达差异。前者的比较可用于筛选可能预防（或促进）白内障形成的潜在候选基因，而后者的比较则有助于解析α3Cx46 KO小鼠白内障形成的分子机制。本实验共使用8个样本，以下4类小鼠各设置2个独立生物学重复：129SvJae野生型、129SvJae α3Cx46 KO、C57BL/6J野生型及C57BL/6J α3Cx46 KO。