Expression profiling following Nrf2D29H or Keap1R554Q expression in mouse embryonic fibroblasts. Expression profiling following Nrf2D29H or Keap1R554Q expression in mouse embryonic fibroblasts

We generated mouse embryonic fibroblasts from genetically engineered mouse models harboring conditional alleles of Nfe2l2(Nrf2)D29H and Keap1R554Q and induced expresison of these mutations using adenoviral Cre. Gene expression was profiled. Overall design: MEFs were isolated at embryonic day 13.5-14.5. Homozygous Nrf2D29H or Keap1R554Q MEFs were infected with empty adenovirus (control) or adenoviral cre to induce expression of the mutations. Keap1 was functionally wild-type in adenoviral mock conditions, and homozygous mutant after Ad-Cre infection. Nrf2 was functionally knock-out prior to infection and in adenoviral-mock cells, and homozygous D29H after infection with adenoviral Cre. MEFs were collected 4 days after infection for RNA.

本研究利用携带Nfe2l2(Nrf2)D29H与Keap1R554Q条件性等位基因的基因工程小鼠模型构建小鼠胚胎成纤维细胞（mouse embryonic fibroblasts, MEFs），并通过腺病毒Cre（adenoviral Cre）诱导上述突变的表达，随后对细胞的基因表达谱进行了检测分析。
总体实验设计如下：
小鼠胚胎成纤维细胞于胚胎发育第13.5至14.5天分离获取。分别以空载体腺病毒（对照组）或腺病毒Cre感染纯合Nrf2D29H或Keap1R554Q MEFs，以诱导对应突变的表达。在腺病毒空白感染（mock）条件下，Keap1呈现功能野生型表型；经腺病毒Cre（Ad-Cre）感染后，Keap1则转变为纯合突变型。感染前及腺病毒空白感染的细胞中，Nrf2处于功能敲除状态；经腺病毒Cre感染后，Nrf2则转变为纯合D29H突变型。于感染后4天收集小鼠胚胎成纤维细胞以提取总RNA，用于后续基因表达谱检测。