Transcription profiling of skeletal muscle from wild type and mdx mice at three time points

In this study, in order to minimize the genetic variability of muscle samples we have used two approaches. First, we have analyzed the gene expression profile from a single skeletal muscle, the medial gastrocnemius (MG), and not from a pool of different muscles which could have different expression profiles. Second, we have performed the temporal gene expression profiling by extracting the MG muscles of the same individual from the both legs at two different times to minimize the inter-individual genetic variability. The MG muscle represent an excellent candidate for biopsy due to its easy accessible by surgery and also because its biopsy is well tolerated by the animals allowing us to perform another later biopsy in the other leg to obtain two MG samples of the same individual at two different times. Moreover, MG muscle is composed of approximately 20-30% type I (red) fibers and 70-80% type II (white) fibers {Ariano MA, 1973}, {Simard C, 1988}, {Zhan WZ, 1992} and therefore is more representative of skeletal muscle tissue in general than a muscle composed exclusively by red or white fibers.The transcript expression profiles in MG muscles from mdx and wild-type mice were analyzed at 3 weeks, 1.5 months and 3 months of life by using the 430 2.0 gene chips from Affymetrix (n=3 for each condition). The differentially expressed transcripts which showed differences ~1.5-fold were obtained by performing three different comparisons: 1) genes differentially expressed in mdx compared with controls at each point in time (additional file 1); 2) temporal analysis of the genes differentially expressed in mdx mice between the three points in time also compared with the variations in control mice (additional file 2); and 3) temporal analysis of the genes differentially expressed in control mice between the three points in time also compared with the variations in mdx mice (additional file 3). The first comparison that we performed, by comparing the gene expression between mdx and control mice at every point in time, was similar to that performed in previous longitudinal studies {Porter JD, 2003}, {Rouger K, 2002}, {Turk R, 2005}. However, the other two comparisons were directed to elucidate the genes that are varying throughout the period of time analyzed in every mice strain, and therefore we obtained on the one hand the genes that vary in mdx mice but not in wild-type, and on the other hand the genes that vary in control animals but remain unchanged in mdx mice between the times analyzed. To present the results in a more comprehensive form, all the genes were classified in seven different categories: Cell adhesion & extracellular matrix; Proteolysis; Muscle structure & regeneration; Inflammation & immune response; Cell signaling & cell communication; Metabolism; and Others/unknown. The resulting genes from our study were classified in their functional categories using information from Affymetrix (www.affymetrix.com) and from the Gene Ontology database accessible in the Jackson Laboratory Mouse Genome Informatics website (www.informatics.jax.org).本研究为尽可能降低肌肉样本的遗传异质性，采用了两种实验策略。其一，我们仅对单一骨骼肌——腓肠内侧肌（medial gastrocnemius, MG）——进行基因表达谱分析，而非使用多份不同肌肉混合样本——这类混合样本可能存在各异的表达特征。其二，我们通过在两个不同时间点采集同一个体双侧腿部的MG肌肉样本，开展时序基因表达谱分析，以进一步降低个体间的遗传异质性。MG肌肉是活检的理想靶组织，原因在于其手术暴露简便，且动物对该活检操作耐受性良好，这使得我们可在后续对另一侧腿部进行活检，从而获取同一个体在两个不同时间点的两份MG样本。此外，MG肌肉约由20%-30%的I型（红肌）纤维与70%-80%的II型（白肌）纤维构成{Ariano MA, 1973}、{Simard C, 1988}、{Zhan WZ, 1992}，因此相较于仅含红肌或白肌纤维的肌肉组织，其更能代表一般骨骼肌的特征。我们采用Affymetrix公司的430 2.0基因芯片，对3周龄、1.5月龄及3月龄的mdx小鼠与野生型小鼠的MG肌肉转录表达谱进行分析（每组设置3个生物学重复）。通过开展三组不同的比较分析，筛选得到表达差异约1.5倍的差异表达转录本：1）各时间点mdx小鼠与对照小鼠间的差异表达基因（附加文件1）；2）对三个时间点间mdx小鼠的差异表达基因进行时序分析，并同时与对照小鼠的基因表达变化进行比对（附加文件2）；3）对三个时间点间对照小鼠的差异表达基因进行时序分析，并同时与mdx小鼠的基因表达变化进行比对（附加文件3）。我们开展的第一组比较分析——即各时间点下mdx小鼠与对照小鼠的基因表达比对——与此前多项纵向研究的结果一致{Porter JD, 2003}、{Rouger K, 2002}、{Turk R, 2005}。而另外两组比较分析则旨在阐明两种小鼠品系在所分析时段内的差异表达基因：由此我们一方面筛选得到仅在mdx小鼠中存在表达变化、而野生型小鼠无显著变化的基因，另一方面筛选得到仅在对照动物中存在表达变化、而mdx小鼠在对应时段内无显著变化的基因。为更全面地呈现研究结果，我们将所有基因划分为7个功能类别：细胞黏附与细胞外基质、蛋白水解、肌肉结构与再生、炎症与免疫应答、细胞信号传导与细胞通讯、代谢，以及其他/未知功能基因。本研究得到的差异基因通过参考Affymetrix官网（www.affymetrix.com）以及杰克逊实验室小鼠基因组信息学网站（www.informatics.jax.org）提供的基因本体（Gene Ontology）数据库信息，完成了功能分类。