Transgene conformation in RA-differentiated and erythroid cells. (A) High GFP spot number, linear fibroblast-like (left) versus low GFP spot number, compact ES-like (right) BAC conformations in RA-differentiated Dhfr 36 or β-globin 8d cells (red, DAPI; green, GFP-LacI) (B) Erythroblasts derived from ES cells after 13 d of differentiation (May-Giemsa staining). (C) 3D DNA FISH of transgene arrays in ES clones β-globin 8d, Dhfr 36, and α-globin1c cells after differentiation to erythroblasts: DAPI (blue), LacO (green), and BAC probes (red). Whole cells are shown in first left panels with enlargements (marked by white boxes) in right panels. Fringes of BAC signals (carets) overlap the edges of DAPI-poor regions but not LacO signal. Overlapping BAC and LacO signals coincide with high-intensity DAPI staining (arrows). (D) Histograms showing degree of colocalization of LacO and BAC FISH signals in individual cells (as measured by Pearson’s correlation coefficients [Rr]) before (black) or after (gray) differentiation to erythroblasts. Dhfr and α-globin BAC transgenes show clear transition from low to high overlap during differentiation. (E) RNA FISH BAC (red) signal and DAPI (blue) in clones β-globin 8d, Dhfr 36, and α-globin 1c after erythroid differentiation. (right) Enlargements of the boxed areas (left) show that RNA signals (carets) coincide with DAPI-poor regions. DAPI signal in A has been γ processed (factor of 0.7). Bars: (A, C, and E) 2 µm; (B) 20 µm.