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Series GSE4718 Query DataSets for GSE4718
Status Public on Apr 27, 2006
Title Improved DNA methylation analysis via enrichment of demethylated cells expressing an X-inactivated GFP transgene
Organism Mus musculus
Experiment type Expression profiling by array
Methylation profiling by array
Summary 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
Overall design Mice used have an X-linked GFP transgene driven by the chicken β-actin promoter and CMV
intermediate early enhancer in a 129/ICR mixed genetic background obtained from The Jackson
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.
Contributor(s) Vallender TW, Lahn BT
Citation(s) 17068962
Submission date Apr 25, 2006
Last update date Feb 11, 2019
Contact name Tammy Vallender
Phone 7738344065
Organization name University of Chicago
Department Human Genetics
Lab Dr. Bruce Lahn
Street address 929 E 57th St W503V
City Chicago
State/province IL
ZIP/Postal code 60637
Country USA
Platforms (1)
GPL1261 [Mouse430_2] Affymetrix Mouse Genome 430 2.0 Array
Samples (6)
GSM106429 fibroblast_unsorted_control_rep1
GSM106430 fibroblast_unsorted_control_rep2
GSM106505 fibroblast_G-negative_5aza_rep1
BioProject PRJNA95607

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