FEAT overexpression in NIH3T3. Mus musculus

To evaluate whether FEAT has cellular functions relevant in oncogenesis, FEAT was overexpressed in NIH3T3 cells, which only weakly express FEAT protein, and the alterations in genome-wide transcriptional profiles were analyzed by microarrays. Overall design: The ORF of human FEAT cDNA was subcloned into the pENTR3C entry vector of the Gateway cloning system (Invitrogen). LR recombination with the pEF-DEST51 vector (Invitrogen) yielded the pEF-DEST51-FEAT plasmid that encodes FEAT driven by the elongation factor 1alpha promoter. NIH3T3 cells were transfected with pEF-DEST51-FEAT using HilyMax (Dojindo Laboratories). After 7 days of selection with 10 µg/ml blasticidin S (Kaken Pharmaceutical), colonies were picked and screened for clones in which FEAT protein was overexpressed. Three clones, FEAT-3, -15, and -20 were used in further studies. The ccdB gene in the pEF-DEST51 vector was deleted to construct the pEF-DEST51-∆ccdB plasmid, which was stably transfected into NIH3T3 cells to obtain control cell lines, named ∆ccdB-1, -2, and -3.

为评估FEAT是否具备与肿瘤发生相关的细胞功能，本研究在仅微弱表达FEAT蛋白的NIH3T3细胞中过表达FEAT，并通过基因芯片分析全基因组转录谱的变化。总体实验设计：将人FEAT cDNA的开放阅读框（Open Reading Frame）亚克隆至Gateway克隆系统（Invitrogen）的pENTR3C入门载体中。通过与pEF-DEST51载体（Invitrogen）进行LR重组反应，获得由延伸因子1α启动子驱动FEAT表达的pEF-DEST51-FEAT质粒。使用HilyMax转染试剂（Dojindo Laboratories）将该质粒转染NIH3T3细胞，经10 μg/ml杀稻瘟菌素S（Kaken Pharmaceutical）筛选7天后，挑取单克隆并筛选FEAT蛋白过表达的细胞株，最终选取FEAT-3、FEAT-15及FEAT-20三个克隆株开展后续实验。此外，本研究构建了缺失ccdB基因的pEF-DEST51-ΔccdB载体，将其稳定转染NIH3T3细胞以获得对照细胞株，分别命名为ΔccdB-1、ΔccdB-2及ΔccdB-3。