RNAseq analysis of gene expression profiles in a mouse model of tuberous sclerosis

We have generated a tuberous sclerosis complex (TSC) mouse model through mouse Gfap-Cre mediated conditional knockout of Tsc1 gene. Tsc1 depletion occurs in most astrocytes and a fraction of upper cortical and hippocampal neurons. The mice develop spontaneous seizures after 10 weeks of age. To determine the effect of Tsc1 deficiency on astrocytes, we compared astrocyte gene expressio profiles between mGfap-Cre:Tsc1CKO and littermate control mice at 4 weeks and 15 weeks of age. At 4 weeks, mGfap-Cre:Tsc1CKO mice showed increased transcription of a few astrocyte genes, including Vim, Amigo2, Thbs4, Slc7a11, Gjb6 and ALDH1L1, and there were not changes in protein products of these genes. At 15 Weeks, mGfap-Cre:Tsc1CKO mice with seizures showed increased transcription of reactive astrocyte genes, including Gfap, Vim, CD44, Sparc, Serpina3n and many others. The transcriptional changes were consistent with enhanced protein levels. Total RNA (RIN>=9.8) was extracted from upper half cortex of somatosensory and parietal cortices of mGfap-Cre+:Tsc1CKO and control mice. mRNAs were enriched using poly-A-pull-down. Library was prepared using the Illumina TruSeq RNA prep kit, and sequenced using an Illumina HiSeq 2000 system with a read length of 100 bases. RTA (Illumina) was used for base calling and bcl2fastq (1.8.4) was applied to covert BCL to fastaq format. The reads were mapped to a mouse reference genome mm9 using Tophat (2.0.4). Differential gene experssion analysis was conducted using DESeq2 pipeline, with a FDR of 0.1, and an adjusted p value cutoff 0.05.本研究通过小鼠Gfap-Cre介导的Tsc1基因条件性敲除，构建了结节性硬化复合物（tuberous sclerosis complex, TSC）小鼠模型。Tsc1基因缺失主要发生于绝大多数星形胶质细胞，以及一小部分皮层上层和海马神经元中。该模型小鼠在10周龄后会出现自发性癫痫发作。 为探究Tsc1缺失对星形胶质细胞的影响，我们分别在4周龄和15周龄时，对mGfap-Cre:Tsc1CKO小鼠与其同窝对照小鼠的星形胶质细胞基因表达谱进行了比较。在4周龄时，mGfap-Cre:Tsc1CKO小鼠的部分星形胶质细胞基因转录水平升高，包括Vim、Amigo2、Thbs4、Slc7a11、Gjb6及ALDH1L1，但这些基因的蛋白产物水平未发生显著变化。在15周龄时，出现癫痫症状的mGfap-Cre:Tsc1CKO小鼠的反应性星形胶质细胞基因转录水平升高，包括Gfap、Vim、CD44、Sparc、Serpina3n等众多基因，且该转录水平变化与蛋白水平上调相一致。 我们从mGfap-Cre+:Tsc1CKO小鼠及对照小鼠的躯体感觉皮层和顶叶皮层的上半部分组织中提取了总RNA，其RNA完整性数（RNA Integrity Number, RIN）≥9.8。采用poly-A亲和下拉法富集mRNA，使用Illumina TruSeq RNA建库试剂盒构建测序文库，并通过Illumina HiSeq 2000测序系统进行测序，测序读长为100碱基。使用Illumina RTA软件进行碱基识别，并通过bcl2fastq（版本1.8.4）将BCL格式文件转换为fastaq格式。 使用Tophat（版本2.0.4）将测序读段比对至小鼠参考基因组mm9；采用DESeq2分析流程进行差异基因表达分析，设置错误发现率（False Discovery Rate, FDR）阈值为0.1，校正后P值截值为0.05。