Dataset features


Application: Gene expression microarray analysis, DNA methylation array analysis
Number of samples: 6
Release date: Apr 27 2006
Last update date: Aug 23 2018
Access: Public
Taxon: Homo sapiens
Dataset link Improved DNA methylation analysis via enrichment of demethylated cells expressing an X-inactivated GFP transgene

Experimental Protocol

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.










Tammy Vallender