Figure 6. Validation of top candidate insulators in a GFP-based plasmid enhancer-blocking assay.

(A) Reporter plasmid. The reporter construct pJC5-4/P4P2K used for the drug-resistant colony assay was modified by replacing the Neo reporter gene with a GFP fluorescent reporter gene as diagrammed. This construct also contains a second expression cassette for Neo transcribed from the constitutive Pgk gene promoter. Insulator candidate and control fragments were inserted upstream and downstream of the GFP expression cassette. (B) Experimental schema. Plasmid constructs were linearized and transfected into K562 cells as for the drug-resistance colony assay, and selected with low level G418 (0.5 mg/mL) in liquid cultures for 7 days. The level of reporter GFP expression was subsequently analyzed by flow cytometry using the indicated gating. (C) Determination of insulator activity. Histograms represent the average ± standard deviation from a total of 4 independent experiments, and are reported as a percentage of the mean fluorescence for the 308 bp Zeo spacer control (set at 100%). * P<0.05 versus spacer control (t-test).