Targeted degradation of sense and antisense C9orf72 RNA foci as therapy for amyotrophic lateral sclerosis and frontotemporal dementia (strand specific RNA-seq). Mus musculus

Purpose: The purpose of this experiment is to identify expression changes after ASO-dependent depletion of mouse C9orf72 in the spinal cord of wild-type C57Bl/6 female mice. Methods: Strand specific RNA-seq was performed using RNAs extracted from spinal cord of C57Bl/6 mice two weeks after intracerebroventricular stereotactic injection of saline (n=3), a control ASO (n=3) or an ASO targeting mouse C9orf72 (n=3). C9orf72 RNA levels were reduced to approximately 30% of control levels in spinal cords from mice treated with the C9orf72 ASO. Results: Statistical comparison of RPKM values between RNAs from C9orf72 and control ASO treated animals or C9orf72 and saline treated samples revealed that only 12 genes were consistently upregulated (defined by P<0.05 adjusted for multiple testing) and 12 genes including C9orf72 were downregulated (defined by P<0.05 adjusted for multiple testing). Conclusions: Only few RNA expression changes were identified in the spinal cord following reduction of C9orf72. Overall design: Use of strand specific RNA-seq to test the consequences of C9orf72 loss of function in mouse spinal cord.

实验目的：本实验旨在探究野生型C57Bl/6雌性小鼠脊髓中，经反义寡核苷酸（ASO，antisense oligonucleotide）介导的小鼠C9orf72敲减后，基因表达的变化情况。实验方法：对C57Bl/6小鼠进行侧脑室立体定位注射，分别给予生理盐水（n=3）、对照ASO（n=3）或靶向小鼠C9orf72的ASO（n=3），于注射后两周提取脊髓组织总RNA，进行链特异性RNA测序（strand-specific RNA-seq）。经靶向C9orf72的ASO处理的小鼠脊髓中，C9orf72的RNA水平降至对照组的约30%。实验结果：对靶向C9orf72的ASO处理组与对照ASO处理组、靶向C9orf72的ASO处理组与生理盐水处理组的RNA样本的每百万reads映射数（RPKM，reads per kilobase per million mapped reads）值进行统计学比较后发现，仅12个基因呈现持续上调（定义为校正多重检验后P<0.05），且包括C9orf72在内的12个基因呈现持续下调（定义为校正多重检验后P<0.05）。实验结论：在小鼠脊髓中敲减C9orf72后，仅检测到极少的RNA表达变化。整体实验设计：采用链特异性RNA测序（strand-specific RNA-seq），探究小鼠脊髓中C9orf72功能丧失所产生的影响。