Figure 1

Characterization of the 743 transgenic ES cell line. A. Phosphorylation/dephosphorylation analysis of Tyr705 in endogenous and transgenic STAT3. After 24 hours of LIF and OHT deprivation cells were cultivated till their homogenization in presence of either LIF or OHT. During LIF or OHT deprivation no changes in the protein expression levels could be detected, but after 24 hrs the Tyr705 residue of both transgenic and WT STAT3 was completely dephosphorylated. 10 minutes after addition of LIF the tyr705 residue of both STAT3 was phosphorylated whereas after addition of OHT complete phosphorylation was obtained only after 6 hrs. B. Dephosphorylation of Tyr705 was analyzed by eliminating LIF or OHT from respectively WT or 743 cells. Kinetics for the dephosphorylation were slower then for the phosphorylation, only after 48 hrs dephosphorylation of WT Tyr705 was complete whereas complete dephosphorylation of STAT3-MER occurred only after 72 hrs. C. Nanog and STAT3 expression levels were tested by RTQ-PCR. Values are normalized with β-actin and the values indicate fold changes compared to the WT. Both WT and 743 ES cell lines expressed Nanog and the transgenic 743 ES cells have an increased Nanog expression compared to WT. D. After injection of 743 in C57BL/6 host blastocysts a 50–60% chimera was generated. Littermates from the crossing of the chimera with a WT FVB/N female generated white littermates, 50% of which were hemizygous for the transgene, indicating germline competence of the 743 cell line.