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Figures
- Fig. 1.
Isolation of MEL cells with large transgene arrays, tagged with lac operator repeats, and carrying β-globin regulatory sequences. (A) The pPALZ8.8 plasmid was created by cloning several parts into pSV2-neo (black and dark green). Dotted blue and green boxes (flanked by ClaI and EcoRI restriction sites) indicate the hypersensitive sites HS1-HS4 of the human β-globin locus which form the μLCR. The human β-globin promoter (filled pink) controls a β-galactosidase gene (LacZ, hollow pink). The light blue box indicates 64 repeats of the lac operator, the binding site for the GFP/lac-repressor fusion protein. Arrows indicate open reading frames of the genes involved. Total size of plasmid is 15 kb. Restriction sites mentioned in the Materials and Methods section are indicated. Sites in brackets had been present in the polylinker and were lost during cloning. (B) Cloning scheme for PALZ39 subclones.
- Fig. 2.
Analysis of chromosomes carrying transgene arrays in different MEL subclones reveals a large transgene insertion and cytogenetic instability. FISH using the plasmid pPALZ8.8 (green in A-F, top; red in G) was carried out on metaphase spreads and counterstained for DNA (blue in A-F, top, and G; white in A-F, bottom, and J). (A) The predominant chromosome in PALZ39E cells. (B) Other forms found in PALZ39E. (C) The predominant chromosome in the PALZ39E subclone A9. (D) Other forms found in the A9 subclone. (E) Original PALZ39M chromosome. (F) PALZ39M chromosome found after more than 25 cell culture passages. (G-J) Part of a metaphase plate from the cell line PALZ39E showing the predominant transgene-carrying chromosome type (bottom) and a normal chromosome 12 (top). Arrows indicate the borders of the transgene array. (G) Multicolor overlay with transgene array in red, paint probe for mouse chromosome 12 in green and DNA counterstain in blue. White lines in the overlay separate individual chromosomes. The gray-scale images for the transgene array (H), the chromosome 12 paint (I) and the counterstain (J) are also shown.
- Fig. 3.
Transcriptional activation is associated with a severalfold increase in transgene array volume. GFP/lac-repressor signals from transgene arrays in PALZ39E cells from an inducible culture. Projections of confocal image stacks are shown. (A) Uninduced X-Gal-negative cells. (B) Induced X-Gal-positive cells. DNA counterstain in blue. Scale bar is for all images.
- Fig. 4.
Volume measurements of transgene arrays in β-galactosidase-expressing (blue dots) and non-expressing (white dots) cells. Each dot represents one nucleus. Dots above each other represent nuclei from the same image stack, thus excluding differences in local specimen conditions or image recording between these nuclei. (A) GFP/lac-repressor signals in PALZ39E cells from an inducible culture. Signals were recorded either from uninduced, non-expressing or induced, expressing cells. (B) FISH signals from an inducible PALZ39E culture that was either not induced (left) or induced with HMBA (right). (C) Uninducible PALZ39E cells. (D) PALZ39M cells.
- Table 1.
Mean values of transgene signal sizes in different cultures
β-Galactosidase positive β-Galactosidase negative Inducible PALZ39E, +HMBA* 2.2 μm3, n=43 n.d. Inducible PALZ39E, no HMBA* n.d. 0.6 μm3, n=26 Inducible PALZ39E, +HMBA‡ 6.4 μm3, n=50 1.5 μm3, n=20 Inducible PALZ39E, no HMBA‡ 6.4 μm3, n=18 1.8 μm3, n=70 Non-inducible PALZ39E‡ 6.2 μm3, n=24 3.3 μm3, n=22 PALZ39M‡ 4.7 μm3, n=23 1.1 μm3, n=49 - Fig. 5.
Increase in transgene array volume associated with transcriptional activation is also observed using FISH. (A) PALZ39E nuclei with FISH signals for the transgene (green) and the centromeres (red). DNA counterstain in blue. Inset shows corresponding X-Gal staining. (B) PALZ39M nuclei with FISH signals for the transgene (green) in PALZ39M cells. Inset shows X-Gal staining. (C-F) Nuclei from PALZ39E cells without induction and X-Gal-negative (C,D) or with induction and X-Gal-positive (E,F). Colors as in (A). Fluorescence images are projections of confocal sections. Scale bar is for all fluorescence images.
- Fig. 6.
Different radial distribution of active (blue) and inactive (gray) transgene arrays within interphase nuclei. Each nucleus was divided into 25 shells of equal thickness. 25 points on each curve show the percentage of the signal (transgene or counterstain) within each shell. The x-axis indicates the position of the center of the shell relative to the nuclear radius (which is defined as 100 units). Detection of the transgene in inducible PALZ39E cells was with GFP/lac-repressor (A; n=40 for expressing cells, n=12 for non-expressing cells) or by FISH (B; n=22 for expressing cells, n=25 for non-expressing cells). Signals in PALZ39M cells (C; n=22 for expressing cells, n=40 for non-expressing cells) were detected by FISH. Distribution of nuclear DNA is shown in red (from nuclei with active transgenes) and pink (from nuclei with inactive transgenes) for comparison. Red and pink curves are very similar, in (C), they completely overlap, so that the latter is not visible. Error bars indicate the standard deviation of the mean.
- Table 2.
Positions of transgene arrays relative to chromocenters (centromere clusters)
A* B* C* D* Sample size Inducible PALZ39E, no HMBA, X-Gal positive 50% 33% 17% 0% 18 Inducible PALZ39E, no HMBA, X-Gal negative 93% 2% 4% 0% 45 Inducible PALZ39E, with HMBA, X-Gal positive 81% 15% 4% 0% 26 Inducible PALZ39E, with HMBA, X-Gal negative 89% 11% 0% 0% 19 PALZ39M, X-Gal positive 78% 17% 4% 0% 23 PALZ39M, X-Gal negative 98% 2% 0% 0% 49 -
↵* A, no contact; B, touching; C, partly overlapping; D, complete colocalization.
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