Nuclear localization of the hCD2 transgene in hCD2+ and hCD2- T cells. (A, B, C) Three-dimensional fluorescence in in situ hybridization (FISH) analysis of the hCD2 transgene location in the nuclei of CD2 1.3A14 and MG4 transgenic T cells. (A) Deconvoluted images showing typical examples of two nuclear localization patterns of the hCD2 transgene (red) relative to γ-satellite clusters (green) and to the nuclear periphery (determined by DAPI-staining, blue). Bar: 5 μm. (B) The distance between the hCD2 transgene signal and the nearest γ-satellite clusters or nuclear periphery in CD2 1.3A14 hCD2+ and hCD2- T cells. The distance was measured from the centre of the transgene signal to the edge of a pericentric cluster or nuclear periphery. Median for hCD2+ T cells = 0.76 μm, that for hCD2- T cells = 0.29 μm. The difference in the distance of the transgene signal from heterochromatin between hCD2+ and hCD2- T cells was statistically significant: P < 0.0001 by K-S test. (C) The percentage of hCD2+ and hCD2- T cells from the indicated transgenic lines that show close proximity of the transgene signal to heterochromatic nuclear compartments. The difference in the transgene location between hCD2+ and hCD2- T cells was statistically significant (P < 0.0001 by χ2 test). Note that as expected the difference in the transgene location between Mg4 and CD2 1.3A14 hCD2+ T cells was not statistically significant (P > 0.05 by χ2 test). (D) The position of the hCD2 locus relative to the centromeric cluster was determined in sorted CD2+ and CD2- T cells, by cryoFISH using Rhodamine-labelled γ-satellite for detection of the centromeric cluster (red) and the DIG-labelled hCD2-cos1 cosmid probe to detect hCD2 loci (green). Nucleic acids were counterstained with TOTO-3 (blue). Bar: 2 μm. (E) The frequency of association of hCD2 locus with centromeric cluster were measured from the centre of the hCD2 signal to the periphery of the γ-satellite signal.