Figure 5.

The insulator function of the ICR can be bypassed in human cancer cells and correlates to levels of PLAG1 expression. (A) Schematic illustrations of the GFP reporter constructs used in the insulator assays. The analysis of GFP expression was performed using flow cytometry (upper right) where the bars depicts relative GFP expression after correlation to RFP expression (pRep9RFP) and with confocal microscopy where the panels show the GFP expression pattern of the different reporter genes in a subset of cells in Hep3B and JEG-3. The left sections show GFP expression in the construct. The middle section of each panel shows the expression of the transfection control construct, DsRed2. The right sections show an overlay, where the distinctive GFP and RFP expression pattern can be determined. (B) The chromatin conformation of the transfected ICR is similar in both GFP expressing and non-expressing cells. DNase I-treated nuclei from JEG-3 and Hep3B transfected with pB-GFP, was digested with StuI. The nuclease hypersensitive sites (NHSS I and II) correspond to CTCF target sites [Kanduri et al(23)] and are identical in both cell lines.