Table 2.
Summary of three mitotic recombination experiments with DT1E9 ESCs
| Experiment 1 | Experiment 2 | Experiment 3 (2i+LlF) | |
|---|---|---|---|
| 10 μm BrdU before Cre | 35 (n.d.) | n.d. | n.d. |
| 1.25 μm BrdU before Cre | 300 (17:3) | 150 (18:0) | 370 (24:0) |
| 1.25 μm BrdU before Cre | n.d. | 150 (16:3) | 225 (22:0) |
| Control | 1,000 (19:0) | 250 (19:0) | 740 (24:0) |
Total numbers of HATR colonies are compared. Subsequent X/Z segregation ratio determined by Southern blot analysis of the initially heterozygous distal Snrpn marker is shown in brackets. Toxicity of 10-μM BrdU in the first experiment had such a profound effect on colony numbers that we did not include it in experiments 2 and 3. Here, only 1.25-μM BrdU concentration was tested and Cre transfection occurred as indicated. We observed low-level emergence (~10 %) of Z segregants at 1.25-μM BrdU in experiments 1 and 2 but not in experiment 3 where ESCs were cultured in differentiation-suppressing 2i+LIF conditions. This may indicate that Z segregants represent differentiated cells, consistent with ESCs’ tendency to differntiate after exposure of DNA damaging agents. Bottom: summary of the experimental workflow of these assays.
n.d. not done