Founder Line | No Of embryos | Medium Supplements | No of ICM picked | No of stem cell colonies | Cell lines | Transgenic |
|---|
WT | 15 | LIF | 8 (53%)* | 5 (62%)** | 5 | - |
| | 15 | OHT | 4 (27%)* | 0 (0%)** | - | - |
741 | 28 | OHT | 23 (82%)* | 10 (43 %)** | 7 | 7 (100 %)# |
743 | 28 | OHT | 14 (50%)* | 10 (71 %)** | 10 | 10 (100 %)# |
741-F1 | 26 | OHT | 18 (69%)* | 8 (44 %)** | 8 | 8 (100 %)# |
- * percent ICM per plated embryo; ** percent colonies per picked ICM
- # percent transgenic per cell lines
- WT FVB/N or hemizygous FVB/N males carrying the STAT3-MER transgene (lines 741 and 743) were mated with WT FVB/N females. Blastocysts were cultivated either in presence of LIF or OHT. Outgrown ICM's were picked and cultivated till ES cell colonies were visible. Cells were tested for the presence of the transgene by PCR analysis. As expected it was possible to establish WT ES cells only in presence of LIF but not when OHT alone was added to the culture. Furthermore in presence of OHT both transgenic lines 741 and 743 generated ES cell colonies that were carrying the transgene. Because theoretically only 50% of the blastocysts were expected to be transgene it is to assume that the establishment frequency was almost 100%. After generation of germline competent chimera with the newly established 741 cell line hemizygous transgenic F1 animals were generated and mated with WT FVB/N females. In this breeding the establishment efficiency in presence of OHT was similar to one observed in the parental blastocysts indicating that the STAT3-MER induced stabilization is kept also in the F1 generation.
