Expression analysis of p53-/- and p53-/-, Ha-RasV12-transformed MEFs upon E4F1 gene inactivation

E4F1 is a ubiquitously expressed zinc-finger protein of the Gli-Kruppel family that was first identified, more than 30 years ago, as a cellular target of the adenoviral oncoprotein E1A13S (Ad type V), required for transcriptional regulation of adenoviral genes. In order to identify the p53-independent program controlled by E4F1, we performed microarray analyses in p53 KO and p53 KO; Ha-RasV12-transformed mouse embryonic fibroblasts (MEFs) in wild type and E4F1-inactivated cells. To address p53-independent E4F1 transcriptome, a 12 chip array study has been realized using total RNA recovered from wild-type (E4F1+/flox, CRE infected; odd Samples) MEFs and E4F1-depleted (E4F1-/flox, CRE infected; even Samples) MEFs in p53-/- (Samples 1 to 12) and p53-/-, Ha-RasV12 background (Samples 13 to 24). p53-/- MEFs were derived from 13.5-day mouse embryos. Transformed p53-/- MEFs were generated by infection with a recombinant retrovirus encoding for Ha-RasV12. Three independent biological replicates of wild-type and knock-out MEFs for E4F1 have been used on the two genetic backgrounds.E4F1是一种广泛表达的Gli-Kruppel家族锌指蛋白，三十余年前首次被鉴定为腺病毒癌蛋白E1A13S（Ad V型）的细胞靶点，该蛋白参与腺病毒基因的转录调控。为鉴定E4F1调控的不依赖p53的基因表达程序，我们分别在p53基因敲除（p53 KO）以及经Ha-RasV12转化的p53基因敲除小鼠胚胎成纤维细胞（mouse embryonic fibroblasts, MEFs）的野生型与E4F1功能失活细胞中开展了基因芯片分析。为解析E4F1调控的不依赖p53的转录组，本研究开展了一项包含12张芯片的阵列分析实验，所使用的总RNA提取自两种遗传背景下的细胞：一是p53纯合敲除（p53-/-，样本1至12）的野生型（E4F1+/flox，经CRE重组酶感染；奇数样本）与E4F1敲除（E4F1-/flox，经CRE重组酶感染；偶数样本）MEFs；二是p53纯合敲除且携带Ha-RasV12（样本13至24）的上述两种基因型MEFs。p53-/- MEFs提取自妊娠13.5天的小鼠胚胎。经Ha-RasV12转化的p53-/- MEFs通过感染携带Ha-RasV12编码序列的重组逆转录病毒构建获得。本研究在两种遗传背景下，均使用了3次独立的野生型与E4F1敲除MEFs生物学重复样本。