Figure 3.

Competition between nuclear peripheral versus chromocenter targeting. (a) D4 and D8D14 BAC transgenes exhibit significantly higher percentages of chromocenter association compared with intact (HBB) or D5 deletion (HBBD5) β-globin BAC transgenes. (b) Representative cells from three independent cell clones showing association of HBBD4 transgenes (green) with chromocenters. Blue, DNA DAPI staining. Bars, 2 µm. The presented images were collected in several different experiments and using different exposure times, as appropriate to each particular cell. (c) Stacked bar plots show a near constant sum of peripheral or chromocenter transgene targeting for multiple, independent cell clones carrying HBB, HBBD4, or HBBD5 transgenes or a mixed population of stably selected cell clones for HBB BAC transgenes not containing LacO repeats, suggesting competition between targeting to the nuclear periphery versus chromocenter. (a and c) Localization of BAC transgenes to either the nuclear periphery or chromocenter was measured in ≥50 cells for each cell clone. (d and e) Quantitative real-time PCR analysis of relative mRNA expression levels, normalized by copy number, for β-globin (HBB), olfactory receptor (OR), and selectable marker (kanamycin/neomycin [KN]) genes in cell clones containing either HBB BAC or HBBD4 BAC. Expression per gene copy was normalized relative to the expression in the cell clone containing HBB BAC (d) or the expression of the endogenous mouse HBB gene (e). Data show means ± SEM from three independent experiments.