Transcription profiling of hippocapus from eight different inbred mouse strains to identify substantial strain-specific variation in hippocampal gene expression

Mouse inbred strains differ in many aspects of their phenotypes, and it is known that gene expression does so too. This gives us an opportunity to isolate the genetic aspect of variation in expression and compare it to other phenotypic variables. We have investigated these issues using an eight-strain expression profile comparison with four replicates per strain on Affymetrix MGU74av2 GeneChips focusing on one well-defined brain tissue (the hippocampus). We identified substantial strain-specific variation in hippocampal gene expression, with more than two hundred genes showing strain differences by a very conservative criterion. Many such genetically driven differences in gene expression are likely to result in functional differences including differences in behaviour. A large panel of inbred strains could be used to identify genes functionally involved in particular phenotypes, similar to genetic correlation. The genetic correlation between expression profiles and function is potentially very powerful, especially given the current large-scale generation of phenotypic data on multiple strains (the Mouse Phenome Project). As an example, the strongest genetic correlation between more than 200 probe sets showing significant differences among our eight inbred strains and a ranking of these strains by aggression phenotype was found for Comt, a gene known to be involved in aggression. Experiment Overall Design: Total hippocampal RNA was extracted from eight inbred strains of mice and pooled within each strain (six mice/pool, one pool/strain). cDNA samples (eight) were prepared from the total hippocampal RNA and cRNA was prepared twice from cDNA. (Samples labelled 1 & 2 for each strain were from the first cRNA synthesis preparation, samples labelled 3 & 4 for each strain were from the second cRNA synthesis preparation).Each set of cRNA samples (16 in total) were then hybridized in duplicate to the Murine Genome U74Av2 Array (Chip A; Affymetrix, Santa Clara, USA). In total, 32 arrays were used; four arrays (two arrays with cRNA sample replicates in duplicate) for each of eight strains.小鼠近交品系（Mouse inbred strains）在诸多表型特征上存在显著差异，而基因表达同样存在此类差异。这为我们分离表达变异的遗传维度，并将其与其他表型变量进行对比提供了绝佳契机。我们采用8个品系的表达谱对比分析开展相关研究，每个品系设置4次生物学重复，实验使用Affymetrix MGU74av2基因芯片（Affymetrix MGU74av2 GeneChips），并聚焦于特征明确的脑组织——海马体。 我们鉴定出海马体基因表达中存在显著的品系特异性变异，依据极为严苛的筛选标准，有超过200个基因呈现出品系间表达差异。此类由遗传因素驱动的基因表达差异，大概率会引发包括行为差异在内的多种功能层面变化。 利用大型近交品系面板，可鉴定出与特定表型功能相关的基因，其研究思路与遗传相关性分析相仿。表达谱与功能间的遗传相关性具备极强的研究潜力，考虑到当前针对多个品系的大规模表型数据生成工作——小鼠表型组计划（Mouse Phenome Project），这一优势尤为突出。 举例而言，在我们的8个近交品系中，200余个存在显著表达差异的探针组，与依据攻击行为表型对这些品系进行的排序之间，最强的遗传相关性出现在Comt基因——该基因已被证实与攻击行为密切相关。 实验整体设计如下：从8个近交品系小鼠体内提取总海马体RNA，每个品系内将样本混合（每混合样本包含6只小鼠，每个品系对应1个混合样本）。以总海马体RNA制备8份cDNA样本，再从每份cDNA样本中分别制备2次cRNA。（每个品系标记为1和2的样本均来自首次cRNA合成，标记为3和4的样本则来自第二次cRNA合成。）随后将全部16份cRNA样本分别进行两次重复杂交至小鼠基因组U74Av2芯片（芯片A；Affymetrix公司，美国圣克拉拉）。本次实验总计使用32张芯片：每个品系对应4张芯片（其中2张芯片对应同一份cRNA样本的重复杂交）。