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Transcription profiling of mouse primary dermal fibroblasts treated with DNA demethylating agent 5-aza-2-deoxycytidine and sorted by X-chromosome-linked GFP expression

ABSTRACT: 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.

ORGANISM(S): Mus musculus

SUBMITTER: Bruce Lahn

PROVIDER: E-MEXP-704 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Improved DNA methylation analysis via enrichment of demethylated cells expressing an X-inactivated transgene.

Vallender Tammy W TW Lahn Bruce T BT

BioTechniques 20061001 4

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 describe a method that uses the reactivation of an X-inactivated green fluorescent protein (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 ...[more]

PMID: 17068962

Similar Datasets

Transcription profiling of mouse dermal fibroblasts from X linked GFP transgenics - improved dna methylation analysis via enrichment of demethylated cells expressing an X-inactivated GFP transgene

Project description:The demethylating drug 5-aza-2â-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 methodâs utility in identifying genes activated by global DNA demethylation. Experiment Overall Design: Mice used have an X-linked GFP transgene driven by the chicken Î²-actin promoter and CMV Experiment Overall Design: intermediate early enhancer in a 129/ICR mixed genetic background obtained from The Jackson Experiment Overall Design: Laboratory. Male transgenic animals were bred to wildtype BL6 female mice to obtain females that were heterozygous for the transgene. Dermal fibroblasts were obtained from explant cultures of neonatal female mice from these crosses. The cells were then sorted using FACS to obtain only GFP-nonexpressing cells. These cells represent the population with the GFP transgene on the inactive X chromosome. The GFP-silent cells were treated with the demethylating drug 5-aza-2'-deoxycytidine. This drug does not affect all cells and uses a passive form of demethylation that results in a heterogenous population of unaffected and demethylated cells. A portion of the cells affected by demethylation activate the inactive GFP transgene by demethylating the inactive X chromosome. The GFP-active and GFP-inactive cells were sorted by FACS and assayed by microarray to determine what genes were affected by demethylation. Also the microarray results indicated if sorting for GFP-active cells enriches for cells affected by the drug.

2008-06-13 | E-GEOD-4718 | biostudies-arrayexpress

Transcription profiling of primary mouse dermal fibroblasts, chondrocytes and osteoblasts treated with 5-aza-2?-deoxycytidine for 96/72h to identify key developmental regulator genes whose expression in terminally differentiated cells may be inhibited by DNA methylation

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.

2007-11-23 | E-GEOD-4768 | biostudies-arrayexpress

Transcription profiling of mouse primary dermal fibroblasts treated with the de-methylating agent 5-aza-2?-deoxycytidine vs untreated to investigate methylation related changes in gene expression

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.

2007-11-23 | E-GEOD-4696 | biostudies-arrayexpress

Transcription profiling of mouse primary dermal fibroblasts treated with DNA demethylating agent 5-aza-2-deoxycytidine

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.

2005-01-01 | E-MEXP-701 | biostudies-arrayexpress

Dll1 marks early secretory progenitors in gut crypts that can revert to stem cells upon tissue damage

Project description:The introduction of GFP into the Dll1 locus has made it possible to isolate and study primary intestinal Dll1 positive cells, which constitute the secretory cell precursors of the intestine. Applying DNA array technology, we profiled the RNA changes of FACS-sorted Dll1_high/CD24_low, Dll1_high/CD24_medium and Dll1_high/CD24_high cells . We used cell fractions of intestines from Dll1-EGFP-ires-CreERT2 mice, expressing GFP under the control of the Dll1 promoter. RNA was isolated from three FACS sorted cell populations: all are expressing Dll1-GFP at high levels, but are expressing different levels of Cd24 (low, medium and high). For this set two biological replicates were generated. One additional fraction was isolated from Lgr5-EGFP-ires-CreERT2 mice, expressing GFP under the control of the Lgr5 promoter. This cell fraction contains intestinal stem cells. Differentially labelled cRNA from these cell fractions was hybridized against a reference (RNA isolated from whole intestine) on 4X44K Agilent Whole Mouse Genome dual colour Microarrays (G4122F).

2012-09-27 | E-GEOD-39915 | biostudies-arrayexpress

Genome-wide maps of Six2 and b-catenin in mesenchymal nephron progenitors

Project description:Self-renewing undifferentiated nephron progenitors express Six2, a transcription factor that is required for their maintenance as undifferentiated progenitors. Differentiation of nephron progenitors is triggered by Wnt/b-catenin signaling. In order to understand how Six2 and Wnt signaling counteract each other, we performed ChIP-seq of Six2 and b-catenin in mesenchymal nephron progenitor cells. Nephron progenitors were FACS-isolated from BAC transgenic Six2GFPcre-positive embryonic kidneys at E16.5. For Six2 ChIP, freshly FACS isolated Six2+ cells were used. For b-catenin ChIP, FACS isolated Six2+ cells were aggregated by centrifugation at 850g for 5min and incubated in 10%FBS/DMEM containing 4uM BIO for 24hrs.

2012-09-11 | E-GEOD-39837 | biostudies-arrayexpress

Streptococcus pyogenes MGAS5005: control vs heme exposure

Project description:Transcriptional profiling of Streptococcus pyogenes MGAS5005 cells comparing control untreated GAS cells with GAS cells exposed to 4uM heme for 1.5 h

2014-09-16 | GSE61415 | GEO

Comparison of gene expression signature between nascent Lhx6-GFP+ cells from mouse embryonic brain and differentiated Lhx6-GFP+ cells from ES cells (as well as comparing those of Lhx6-GFP+ and Lhx6-GFP- cells from ES cells)

Project description:There was a remarkable similarity in the molecular properties of the MGE-GFP+ and ES-GFP+ cells. In particular, genes that are important for medial ganglionic eminence (MGE) and cortical interneurons development are both high in expression in both MGE-Lhx6-GFP+ and ES-Lhx6-GFP+ cells (compared to ES-Lhx6-GFP- cells). To investigate how closely ES cells-derived Lhx6-GFP+ cells resembled authentic Lhx6+ MGE cells, and to define the molecular properties of the Lhx6-GFP+ and Lhx6-GFP- cells from differentiated ES cells, we compared their gene expression profiles. We used FACS to purify GFP+ cells from the E12.5 MGE of Lhx6-GFP transgenic mice. ES-Lhx6-GFP+ cells and ES-Lhx6-GFP- cells (both from D12 EB aggregates) were also isolated by fluorescent activated cell sorting (FACS) and all of the RNA samples were subjected to RNA expression microarray analyses.

2013-01-16 | E-GEOD-43508 | biostudies-arrayexpress

Mus musculus

Project description:Murine Kidney Papilla Cells: Control GFP- vs. GFP+ label retaining cells. Cell populations were separated by FACS.

| PRJNA291828 | ENA

Proteomics of FACS-sorted heterogeneous Corynebacterium glutamicum populations

Project description:The metabolic status of individual cells in microbial cultures can differ being relevant for biotechnology, environmental and medical microbiology. However, it is hardly understood in molecular detail due to limitations of current analytical tools. Here, we demonstrate that FACS in combination with proteomics can be used to sort and analyze cell populations based on their metabolic state. A previously established GFP reporter system was used to detect and sort single Corynebacterium glutamicum cells based on the concentration of branched chain amino acids (BCAA) using FACS. A proteomics workflow optimized for small cell numbers was used to quantitatively compare proteomes of a ?aceE mutant, lacking functional pyruvate dehydrogenase (PD), and the wild type. About 800 proteins could be quantified from 1,000,000 cells. In the ?aceE mutant BCAA production was coordinated with upregulation of the glyoxylate cycle and TCA cycle to counter the lack of acetyl CoA resulting from the deletion of aceE.

2017-11-02 | PXD005812 | Pride

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