Comparison of adult intact naive C57Bl/6 Nogo-A KO versus WT spinal cord

Nogo-A localized on myelin adaxonal membrane in the adult CNS is well known for its role as neurite outgrowth inhibitor following a lesion. Nogo-A KO mice show enhanced regenerative/compensatory fiber growth following CNS lesion. However, changes undergoing in their intact CNS have not been studied. Moreover, Nogo-A in the intact adult CNS in also expressed in some neuronal subpopulations, e.g. in the hippocampus, olfactory bulbs and dorsal root ganglia. We compared the intact adult CNS (spinal cord) of Nogo-A KO mice in order to identify: potential compensating molecules which could be interesting new inhibitory neurite outgrowth candidates, possible molecules involved in the up to now not yet clarified downstream signalling pathway of Nogo-A, additional new functions for myelin or neuronal Nogo-A in the intact adult CNS. Keywords: gene expression, Nogo-A KO, spinal cord, adult, naive, unlesioned Spinal cords from 3 adult C57Bl/6 wild type and Nogo-A KO mice have been explanted. Total RNA has been extracted and processed for hybridization on Mouse 430 2.0 Affymetrix GeneChips. Following scanning and first analysis with MAS 5.0, further analysis was performed by GeneSpring 7.2 (Silicon Genetics, Redwood City, CA). A present call filter (2 out of 3 present calls in at least one out of the different studied conditions) was applied. Normalization was run per chip as well as per gene to the median of the control replicates. Data were statistical restricted through a 1-way Anova (p=0.05). A final threshold of =1.2 folds of increase or decrease in the expression level of each single transcript was applied. Regulated transcripts have been assigned to functional categories according to GeneOntology as well as literature and database mining (Pubmed and Bioinformatics Harvester EMBL Heidelberg).已知定位于成年中枢神经系统（CNS）髓鞘轴突膜的Nogo-A，是中枢神经系统损伤后经典的神经突生长抑制因子。Nogo-A敲除（KO）小鼠在中枢神经系统损伤后，其再生/代偿性纤维生长能力显著增强。然而，该类小鼠完整中枢神经系统内的固有变化尚未得到研究。此外，完整成年中枢神经系统中的Nogo-A还在部分神经元亚群中表达，例如海马体、嗅球与背根神经节。本研究以Nogo-A敲除小鼠的完整成年中枢神经系统（脊髓）为研究对象，通过比较分析旨在实现三大目标：一是筛选潜在的补偿性分子，此类分子有望成为新型神经突生长抑制候选靶点；二是挖掘迄今尚未阐明的Nogo-A下游信号通路相关分子；三是揭示完整成年中枢神经系统内髓鞘源性或神经元源性Nogo-A的全新功能。 关键词：基因表达、Nogo-A敲除（Nogo-A KO）、脊髓、成年、未损伤 本研究采集3只成年C57BL/6野生型小鼠与3只Nogo-A敲除小鼠的脊髓组织。提取总RNA后，在小鼠430 2.0型Affymetrix基因芯片上完成杂交实验。芯片扫描并通过MAS 5.0完成初步数据分析后，采用GeneSpring 7.2软件（Silicon Genetics公司，美国加利福尼亚州雷德伍德市）进行后续分析。设置存在性呼叫过滤条件：至少在一种研究分组的3次生物学重复中，需满足2次及以上阳性呼叫。以对照组重复样本的表达量中位数为参照，分别对单张芯片与单个基因进行标准化处理。通过单因素方差分析（1-way ANOVA，p=0.05）对数据进行统计学筛选。最终设置差异表达阈值为单个转录本的表达水平上调或下调幅度不低于1.2倍。根据基因本体（GeneOntology）、相关文献及公开数据库（PubMed与欧洲分子生物学实验室海德堡生物信息学采集器（Bioinformatics Harvester EMBL Heidelberg）），将差异表达转录本注释至相应功能类别。