Project description:DNA methylation can contribute to the stable transcriptional silencing of mammalian genes. Oftentimes, these genes are important developmental regulators, and their silencing in cell types where they are not supposed to be active is important for the phenotypic stability of the cells. To identify key developmental regulator genes whose expression in terminally differentiated cells may be inhibited by DNA methylation, mouse dermal fibroblasts were demethylated with 5-aza-2-deoxycytidine, and changes in gene expression monitored by microarray analysis. Three biological replicates for both control and 5-aza-2'-deoxycytidine treatment were derived. Cells were primary mouse dermal fibroblasts, primary mouse chondrocytes, and primary mouse osteoblasts derived separately for each biological replicate. Treated cells were exposed to 5uM 5-aza-2'-deoxycytidine for 96 hours with recovery in normal medium for 24 hours. Control cells were untreated.
Project description:DNA methylation can contribute to the stable transcriptional silencing of mammalian genes. Oftentimes, these genes are important developmental regulators, and their silencing in cell types where they are not supposed to be active is important for the phenotypic stability of the cells. To identify key developmental regulator genes whose expression in terminally differentiated cells may be inhibited by DNA methylation, mouse dermal fibroblasts were demethylated with 5-aza-2-deoxycytidine, and changes in gene expression monitored by microarray analysis. Three biological replicates for both control and 5-aza-2'-deoxycytidine treatment were derived. Cells were primary mouse dermal fibroblasts derived by explant procedure separately for each biological replicate. Treated cells were exposed to 5uM 5-aza-2'-deoxycytidine for 96 hours with recovery in normal medium for 24 hours. Control cells were untreated.
Project description:DNA methylation can contribute to the stable transcriptional silencing of mammalian genes. Oftentimes, these genes are important developmental regulators, and their silencing in cell types where they are not supposed to be active is important for the phenotypic stability of the cells. To identify key developmental regulator genes whose expression in terminally differentiated cells may be inhibited by DNA methylation, mouse dermal fibroblasts were demethylated with 5-aza-2’-deoxycytidine, and changes in gene expression monitored by microarray analysis. Keywords: 5-aza-2'-deoxycytidine; methylation; dermal fibroblast
Project description:DNA methylation can contribute to the stable transcriptional silencing of mammalian genes. Oftentimes, these genes are important developmental regulators, and their silencing in cell types where they are not supposed to be active is important for the phenotypic stability of the cells. To identify key developmental regulator genes whose expression in terminally differentiated cells may be inhibited by DNA methylation, mouse dermal fibroblasts were demethylated with 5-aza-2’-deoxycytidine, and changes in gene expression monitored by microarray analysis. Keywords: 5-aza-2'-deoxycytidine; methylation; dermal fibroblast; chondrocyte; osteoblast
Project description:Primary mouse dermal fibroblasts from heterozygous female X chromosome-linked GFP mice were sorted through FACS for GFP non-expressing cells (~50% of cells due to X-inactivation). The GFP- cells were treated with 4uM 5-aza-2'-deoxycytidine (5A2dC) for 48 hours with recovery in normal medium for 24 hours. Following the treatment, cells were again sorted by FACS into GFP-activated and GFP-nonexpressing cells. These populations, along with a control untreated population were assayed for transcriptional changes by microarray.
Project description:Mouse primary dermal fibroblasts were treated with DNA demethylating agent 5-aza-2'-deoxycytidine. The exposure was 5um 5A2dC for 96 hours followed by recovery in normal medium for 24 hours. Three biological replicates were created for both the control and treated populations. Each biological replicate represents a separate derivation of dermal fibroblasts from genetically identical mouse pups aged 0-3 days. Cells reached passage 4 before initiating drug exposure.
Project description:The demethylating drug 5-aza-2’-deoxycytidine (5-aza-2dC) is frequently used to investigate the effect of global DNA demethylation on gene expression in cultured mammalian cells. Here, we utilize a novel method that uses the reactivation of an X-inactivated GFP transgene as a marker to enrich for cells that have undergone drug-induced demethylation. By combining it with microarray gene expression profiling, we demonstrate the method’s utility in identifying genes activated by global DNA demethylation. Keywords: 5-aza-2'-deoxycytidine; methylation; dermal fibroblast; X-linked GFP