Transcription profiling by array of mice overexpressing a transgenic mutant human TDP-43 (hTDP-43) gene and wild-type mice to study TDP-43 mutations in relation to amyotrophic lateral sclerosis

Mutations in TDP-43 (an RNA binding protein) are known to cause amyotrophic lateral sclerosis (ALS). Previously, our group and several other studies showed that TDP-43 binds to several RNA targets in the mammalian CNS. ALS causing mutations in the C-terminal region of TDP-43 that is involved in splicing regulation may lead to aberrant splicing of several RNA transcripts. The main aim of this study is to identify the effect of an ALS causing mutation on the splicing regulation of previously known targets of TDP-43. Mice overexpressing a mutant human TDP-43 (hTDP-43) gene were obtained from The Jackson Laboratory (strain name - B6;CB-Tg(Prnp-TARDBP*A315T) 95Balo/J; Stock no:010700). Mutant hTDP43 was expressed under the control of a prion protein promoter that drives the expression mainly in the mouse CNS and the male transgenic mice developed symptoms around 12 weeks of age while the female mice develop symptoms approximately 20 weeks of age. Therefore, Tg animals that are 50 days old were considered pre-symptomatic and 100 days old (Tg) animals were considered to be in the post-symptomatic stages of the disease for exon array experiments. Age and sex matched pre and post-symptomatic transgenic animals and their wild type littermates were euthanised by cervical dislocation and tissues (brain and spinal cord) were harvested immediately for total RNA isolation experiments (n=3 per group). 200 ng of total RNA from transgenic TDP-43 mice and wild type animals was converted to cDNA and then amplified using the Applause WT-Amp Plus ST kit (NuGEN). Amplified cDNA was then fragmented and biotin labeled using the Encore Biotin Module kit (NuGEN) according to the manufacturer's instruction manual. Labelled cDNA was then hybridised onto Affymetrix GeneChip Exon 1.0 ST arrays in a hybridisation oven at 45 degrees C for 20 hours at 60rpm. After hybridization, washing and staining of the arrays were carried out using Affymetrix fluidics station 450 followed by scanning using GeneChip scanner. GeneChip Command Console Software (AGCC) controlled both the fluidics station and the scanner. CEL files generated by AGCC were uploaded onto Partek software and the data analysis was carried out using the exon array analysis workflow. Exon 1.0 ST arrays contain many more probes, which are classified into three major types based on their source. They are Core, extended and full probe set annotation. Core annotation refers the probe sets that are the most reliable of the three and is derived based on evidences from Refseq and GenBank. Extended annotations refer to probe sets that are generated based on EST sequences, ENSEMBL gene collections and other databases including those used for generating core probe sets. The full annotation refers to probe sets that are purely based on computational predictions. Core probe set annotation, unlike the extended or full annotation excludes the speculative probes reducing the incidence of false positives and was employed for exon array analysis. Alt-splice Anova, a statistical tool available in Partek was used for identifying novel alternative splicing events.TDP-43（RNA结合蛋白，RNA binding protein）的突变已被证实会引发肌萎缩侧索硬化症（amyotrophic lateral sclerosis，ALS）。此前，本课题组及其他多项研究均证实，TDP-43可结合哺乳动物中枢神经系统（central nervous system，CNS）内的多种RNA靶标。引发ALS的TDP-43 C端区域突变参与剪接调控过程，此类突变可诱发多种RNA转录本发生异常剪接。本研究的核心目标是明确此类ALS致病突变对TDP-43已知RNA靶标剪接调控的影响。本研究使用的过表达突变型人源TDP-43（human TDP-43，hTDP-43）基因小鼠购自杰克逊实验室（The Jackson Laboratory），品系为B6;CB-Tg(Prnp-TARDBP*A315T) 95Balo/J，货号：010700。突变型hTDP-43的表达受朊蛋白启动子调控，该启动子主要驱动基因在小鼠中枢神经系统中表达。雄性转基因小鼠约在12周龄时出现疾病症状，而雌性转基因小鼠则约在20周龄时发病。因此，在外显子芯片实验中，我们将50日龄的转基因小鼠视为症状前期个体，100日龄的转基因小鼠则视为疾病症状后期个体。选取年龄与性别相匹配的症状前期、症状后期转基因小鼠及其野生型同窝仔鼠，通过颈椎脱臼法实施安乐死，并立即采集脑组织与脊髓组织用于总RNA提取实验（每组n=3）。取转基因TDP-43小鼠与野生型小鼠的总RNA各200 ng，反转录为互补脱氧核糖核酸（complementary DNA，cDNA）后，采用Applause WT-Amp Plus ST试剂盒（NuGEN公司）进行扩增。按照Encore Biotin Module试剂盒（NuGEN公司）的操作手册说明，对扩增得到的cDNA进行片段化与生物素标记。将标记完成的cDNA置于Affymetrix GeneChip Exon 1.0 ST芯片上，在45℃、60 rpm条件下于杂交炉中杂交20小时。杂交完成后，使用Affymetrix流体工作站450对芯片进行洗涤与染色，随后通过GeneChip扫描仪完成扫描。GeneChip命令控制台软件（GeneChip Command Console Software，AGCC）可同时控制流体工作站与扫描仪。将AGCC生成的CEL文件（CEL file）上传至Partek软件，采用外显子芯片分析工作流完成数据分析。Exon 1.0 ST芯片包含大量探针，根据探针来源可分为三大类别：Core、extended与full探针集注释。Core注释（Core annotation）是三类探针集中可靠性最高的一类，其构建依据来自Refseq与GenBank的实验证据。Extended注释（Extended annotations）指基于EST序列、ENSEMBL基因集以及其他数据库（包括用于构建核心探针集的数据库）生成的探针集。Full注释（full annotation）则指完全基于计算预测得到的探针集。与Extended注释或Full注释不同，Core注释会排除推测性探针，从而降低假阳性发生率，因此本研究的外显子芯片分析采用Core注释方案。采用Partek软件内置的统计工具Alt-splice Anova识别新型可变剪接事件。