Insulator functions of the α-spectrin gene 183-bp exon 1′ + intron 1′ region. (A) Analysis of barrier-element function. The indicated constructs were cotransfected into K562 cells along with a pRSV-neomycin plasmid for selection of individual clones. Individual clones were isolated and expanded in G418 containing medium and switched to nonselective medium before analysis of GFP expression by FACS. The total number of clones analyzed and the number of GFP-expressing (+) clones are shown at the right. Southern blot analysis was used to confirm that all clones had an intact GFP construct. χ2 analysis of the observed versus expected number of GFP+ clones demonstrated that gene silencing was significantly inhibited (P < .001) for all test fragments except for α-spectrin intron 1′ (P > .05). Abbreviations are: HS2: Hypersensitive site 2 from the mouse β-globin cluster locus control region; EGFP: the coding sequence for the enhanced green fluorescent protein gene; cHS4: Hypersensitive site 4 from the chicken β-globin cluster locus control region; ASp: α-spectrin; asterisks denote mutation of the exon 1′ GATA site that disrupts GATA binding to DNA. (B) Analysis of enhancer blocking activity. The indicated constructs were transfected into K562 cells and plated in semisolid medium to allow growth of individual clones. The relative number of colonies was normalized to HS2 γ-Neo. The cHS4 insulator element was used as positive control. HS2, Hypersensitive site 2 from the mouse β-globin gene locus control region; γ, the human γ-globin promoter; Neo, the coding sequence for the neomycin-resistance gene. Error bars represent numbers of normalized colonies obtained from multiple experiments.
