Classification of IRF3- vs ISGF3-dependent gene induction in murine fibroblasts

To investigate the dependency on gene expression on the PRR-activated transcription factor IRF3 versus the type I IFN receptor (IFNAR)-activated transcription factor ISGF3, we extracted primary murine embryonic fibroblasts (MEF) of wild-type, IRF3-knockout or IFNAR-knockout mice (strain C57BL/6J, protocol of Podlech et al., 2002). We stimulated the 3 different cell lines by transfection of immunostimulatory DNA (ISD, 4 hours) for activation of IRF3, or by treatment with murine IFNbeta (3 hours) for activation of the IFNAR-ISGF3 cascade. Mock-transfected (4 hours) or untreated cells served as respective controls. In the last 2 hours before harvest, cells were additionally treated with 200µM 4-thiouridine (4sU) for metabolic labelling of nascent transcripts, and total mRNAs were processed accordingly by SLAM-sequencing procedures for gene expression profiling of newly synthesized transcripts. Overall design: Comparative gene expression profiling analysis of nascent transcripts from SLAM-seq data for gene induction by PRR-IRF3 vs. by IFNAR-ISGF3 signalling.

为探究基因表达对模式识别受体（Pattern Recognition Receptor, PRR）激活的转录因子IRF3与I型干扰素受体（Type I Interferon Receptor, IFNAR）激活的转录因子ISGF3的依赖特征，我们提取了C57BL/6J品系野生型、IRF3敲除及IFNAR敲除小鼠的原代小鼠胚胎成纤维细胞（murine embryonic fibroblasts, MEF），实验方案参照Podlech等人2002年的研究方法。我们通过两种方式分别刺激这3组细胞：一是转染免疫刺激性DNA（immunostimulatory DNA, ISD），刺激4小时以激活IRF3；二是用小鼠β干扰素（murine IFNβ）处理3小时，以激活IFNAR-ISGF3信号级联反应。以空载体转染（4小时）或未处理细胞作为各自的对照。在收获细胞前的最后2小时，我们额外以200μM 4-硫尿苷（4-thiouridine, 4sU）处理细胞，对新生转录本进行代谢标记；随后通过SLAM测序（SLAM-seq）流程处理总mRNA，以实现新生转录本的基因表达谱分析。整体实验设计：基于SLAM-seq数据的新生转录本基因表达谱比较分析，用于对比PRR-IRF3与IFNAR-ISGF3信号通路介导的基因诱导差异。