Fig. 1. Generation of ESCs with inducible exogenous Sox17 expression. (A) Schematic representation of the knock-in strategy of the inducible Sox17 expression cassette into the ROSA26 (R26) locus. The inducible Sox17 expression cassette was inserted into the XbaI site in the ROSA26 first intron. This cassette consisted of a splice acceptor (SA), floxed promoter-less neomycin-resistance gene (neo), tTA, insulator derived from the chicken -globin locus, tTA-responsive human cytomegalovirus minimal promoter (hCMV^*1), rabbit -globin second intron, mouse full-length Sox17 cDNA, internal ribosome entry site (IRES) and EGFP cDNA. Diphtheria toxin A (DTA) cDNA driven by the mouse phosphoglycerate kinase 1 promoter (pPGK) was placed at the 3' end of the targeting construct for negative selection. After the targeting of the ROSA26 locus, neo was excised by the transient expression of Cre recombinase. The locations of the PCR primers used to detect the exogenous Sox17 transcript (primer F and primer R) are indicated. (B) RT-PCR analysis of exogenous Sox17 expression in ESCs grown in the presence or absence of Tet. The black arrowhead denotes the predicted size of the RT-PCR product (2.1 kb). Notice that this size is 0.5 kb less than that of the product derived from genomic DNA as a template (gray arrowhead). Actb is shown as the loading control. (C) Western blotting of the Sox17 protein. The cell lysates of ESCs grown with or without Tet were loaded.