mRNA sequencing analysis of transcripts from hippocampus of Wt1delta mice

Purpose: Study the differentially expressed genes in the hippocampus of mice, comparing Wt1delta mice with their control wild type littermates in basal conditions (naive untrained animals). Methods: Forebrain-specific deletion of Wt1 was achieved by crossing animals homozygous for the conditional Wt1 knockout allele (Wt1fl/fl) with a transgenic line, Camk2a-Cre, (B6.Cg-Tg(Camk2a-cre)T29-1Stl/J; Jackson Lab: http://jaxmice.jax.org/strain/005359.html). Expression of Cre recombinase resulted in the in-frame deletion of exons 8 and 9 and generated a truncated allele encoding a shortened non functional WT1 protein lacking zinc fingers 2 and 3 in their forebrain (starting at P21). Brains of Wt1delta mice (n=2) and their control littermates (wild type; n=2) were removed, dorsal hippocampi were dissected and used for library preparation. The seq library was prepared using TruSeq RNA Sample Prep Kit v2 protocol (Illumina, CA, USA). For the RNA-Seq data analysis Tophat 2.0.13, bowtie 2.1.0 , samtool 0.1.7 and cufflinks 1.3.0 were used. The rn5-bowtie2 index was generated with the command 'bowtie2-build rn5.fa rn5'. The 'rn5.fa'-file was downloaded from the UCSC genome browser. The mm10-bowtie2 index was downloaded from http://bowtie-bio.sourceforge.net/bowtie2/manual.shtml. RefSeq geneTracks and GTF-files for the rn5 and mm10 genome assembly were downloaded from UCSC genome browser. Common gene ids in the GTF-files were matched to individual transcript_ids using the corresponding official symbols obtained from the geneTracks files. Conclusions: Our study illustrates the nature of the different transcripts in Wt1delta mice compared to their control wild type littermates at basal state (untrained). This experiment allowed us to identify wich genes are modulated in the hippocampus by the transcription fator WT1. mRNA profiles from dorsal hippocami Wt1delta mice and control wild type littermates were generated by deep sequencing using TruSeq RNA Sample Prep Kit v2 protocol (Illumina, CA, USA).研究目的：探究小鼠海马体（hippocampus）中的差异表达基因（differentially expressed genes），在基础状态（未经过训练的正常动物）下比较Wt1delta小鼠与其野生型同窝对照小鼠。 研究方法：通过将条件性Wt1敲除等位基因（conditional Wt1 knockout allele）纯合子（Wt1fl/fl）与转基因品系（transgenic line）Camk2a-Cre（B6.Cg-Tg(Camk2a-cre)T29-1Stl/J；杰克逊实验室：http://jaxmice.jax.org/strain/005359.html）杂交，实现前脑（forebrain）特异性Wt1敲除。Cre重组酶（Cre recombinase）的表达会导致第8、9外显子（exons）发生框内缺失（in-frame deletion），从而产生截短等位基因（truncated allele），该基因编码缩短的无功能WT1蛋白，该蛋白在前脑中缺失锌指结构域（zinc fingers）2和3，相关修饰始于出生后第21天（P21）。分别获取2只Wt1delta小鼠（n=2）和2只同窝野生型对照小鼠（n=2）的脑组织，剥离背侧海马体（dorsal hippocampi）用于文库构建（library preparation）。测序文库采用TruSeq RNA Sample Prep Kit v2试剂盒（Illumina，美国加利福尼亚州）按照说明书制备。RNA测序（RNA-Seq）数据分析使用了Tophat 2.0.13、bowtie 2.1.0、samtool 0.1.7及cufflinks 1.3.0。使用命令`bowtie2-build rn5.fa rn5`生成rn5-bowtie2索引，其中`rn5.fa`文件从UCSC基因组浏览器（UCSC genome browser）下载。mm10-bowtie2索引从http://bowtie-bio.sourceforge.net/bowtie2/manual.shtml下载。rn5和mm10基因组组装（genome assembly）的RefSeq基因轨道（geneTracks）及GTF文件（GTF-files）均从UCSC基因组浏览器下载。将GTF文件中的通用基因ID与从geneTracks文件获取的对应官方基因符号（official symbols）匹配至各自的转录本ID（transcript_ids）。 研究结论：本研究阐明了基础状态（未经过训练）下，Wt1delta小鼠相较于野生型同窝对照小鼠的不同转录本特征。本实验使我们得以鉴定转录因子WT1（transcription factor WT1）在海马体中调控的基因。通过TruSeq RNA Sample Prep Kit v2试剂盒（Illumina，美国加利福尼亚州）进行深度测序（deep sequencing），获得了Wt1delta小鼠背侧海马体及野生型同窝对照小鼠的mRNA表达谱（mRNA profiles）。