Attenuation of LMO2 activation by a chromatin insulator (cHS4). (A) The R-6 clone was transduced with scAAV-Cre and transfected with a plasmid containing the LTR-GFP cassette flanked by 1.2-kb cHS4 or 2 × 250-bp core insulator fragments, and single cell clones were identified in which cassette exchange had occurred. The relative LMO2 expression in those clones was compared by qRT-PCR. Both flanking insulators reduced LMO2 expression 3- to 15-fold over that observed in clones having the noninsulated LTR-GFP cassette. (B) Western blot confirmed reduction of LMO2 protein expression by flanking insulator fragments in 6 clones in which the 1.2-kb cHS4 insulator flanked the LTR-GFP cassette relative to the G-25 and R-6 clones. Jurkat cells served as a negative control. (C) The relative GFP expression in clones with or without insulator elements flanking the LTR-GFP cassette was compared by FACS each week for 4 weeks. The average MFI of GFP expression in each clone set is represented by a horizontal bar. LTR-GFP cassette (○), LTR-GFP cassette flanked by a 1.2-kb insulator fragment (●), LTR-GFP cassette flanked by 2 × 250-bp core insulator fragments (▵).