1. Volume 15, Issue 4, Pages 810-817 (April 2007)
Real-time Fluorescence Tracking of Dynamic Transgene Variegation in Stem Cells 
John Ramunas, Heather J Montgomery, Liam Kelly, Tanya Sukonnik, James Ellis, Eric J Jervis 
Molecular Therapy 
Volume 15, Issue 4, Pages (April 2007)
DOI: /sj.mt
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

2. Figure 1
First and last image pairs from tracking experiment. (a) First DIC image (left) and fluorescence image (right) of the time course for colony 1 showing the seven founder cells (time=0 h). Colored disks distinguish cells in different lineages. Founder cells 1 (blue), 3 (yellow), 5 (purple), and 7 (turquoise) were classified as non-fluorescent (brightness <2 SD, or 25±4% in the case of colony 1, above background fluorescence signal), while founders 2 (magenta), 4 (cyan), and 6 (brown) were fluorescent. The sphere (arrow) is one of the 4.7 μm polystyrene beads that separate the confining glass surfaces to form the cell culture space. Fluorescence was measured by averaging the pixel values in a 16×16 pixel (4.7×4.7 μm) square in each cell. (b) DIC image (left) and fluorescence image (right) of the colony at 102.6 h with the colored disks distinguishing the different cell lineages. Supplementary Movie M1 shows the complete set of labeled fluorescence images.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

3. Figure 2
EGFP expression does not affect cell proliferation. (a) Histogram of fluorescence intensity of cells from colonies 1, 2, and 3 that divided (open bars) and died (shaded bars) showing that both populations are bimodal and that bright cells are not over-represented among cells that died, suggesting limited EGFP-mediated phototoxicity. Cells that maintained an average fluorescence of <2 SD above the background fluorescence signal were defined as silent (dashed line). (b) Scatter plot showing the relationship between lifespan of a cell and fluorescence. Among cells that divided in colonies 1 (black squares), 2 (red diamonds), and 3 (blue triangles), fluorescence intensity did not correlate with abnormal lifespan, again indicating limited EGFP-mediated phototoxicity. Fluorescence is reported as a percentage of the range between the dimmest and brightest cells in each of the time courses of the three colonies studied.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

4. Figure 3
Representative expression lineage trees of four lineages from colony 1. (a) The progeny of founder cell 2 silenced over the time-course of the experiment. (b) Lineage 4 expressed the EGFP transgene. (c) Lineage 3 remained silent. (d) The progeny of founder cell 6 bifurcated into two distinguishable transgene expression trajectories. The diameter of the black disks is proportional to the fluorescence intensity of a cell in a given fluorescence image.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

5. Figure 4
The dynamics of transgene variegation is shown by plotting lineage trees sideways, with each branch plotting its own fluorescence while maintaining the connectivity of the tree. Moving averages were applied to the fluorescence levels to remove noise. (a) Lineage 2 was simultaneously silenced around hour 70 of the culture. (b) Lineage 3 remained silent throughout most of the time course. (c) An increase in the heterogeneity of lineage 4 was observed with time. (d) The sublineages of lineage 6 bifurcated.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

6. Figure 5
Inheritance of transgene variegation demonstrated by comparing each cell's average fluorescence to its (a) sister's, (b) mother's, and (c) grandmother's. The degree of correlation between fluorescence intensities is greatest for sister cell pairs (R2=0.93, n=250), less for mother–daughter pairs (R2=0.72, n=250), and even less for grandmother–granddaughter pairs (R2=0.57, n=241). Each color represents cell pairs from lineages 1 to 6 of colony 1 (see Figure 1).
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

7. Figure 6
Representative J1-107 embryonic stem cell lineages after treatment of the founder cell with 5-AzaC. (a) An increase in the fluorescence intensity of all progeny cells was observed. (b) Silent founder cells also expressed the EGFP transgene after treatment with 5-AzaC (increase in fluorescence intensity from below to above 2 SD of the average fluorescent intensity).
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions