FIG. 7.

Double-insulator elements prevented unbalanced expression of transgenes in cis. Uninsulated and insulated dual-reporter plasmids pJTI-R4-EF1α-GFP-EF1α-RFP and pJTI-R4-EF1α-GFP-(cHS4)₂-EF1α-RFP were used to transfect HEK293 cells and the expression level of GFP and RFP was compared (A). Expression of GFP and RFP was biased with GFP being expressed at a much higher level (D–F) in uninsulated clones. However, when 2 copies of cHS4 were placed between these 2 cassettes, expression of GFP and RFP was almost identical as revealed by direct fluorescence microscopy of the native signals (G–I). This was also confirmed by retargeting to the platform hESC line generated in an abnormal hESC line BG01V (J–L) and a widely used normal hESC line WA09 using an episomal expression backbone (M–O). In addition to immunocytochemistry assays, flow cytometric analysis also confirmed that similar percentages of cells expressed both reporters (∼93% for GFP+, as shown in B, ∼90% for TagRFP+, shown in C). x-axis represents the intensity of GFP or TagRFP; y-axis represents the relative number of cells. Nonengineered ESCs were used as negative controls (black curves in B and C). EG:EF1α-EmGFP, ER: EF1α-RFP. EmGFP, emerald green fluorescent protein. Color images available online at www.liebertonline.com/scd