Fig. 2. E-cadherin-specific expression is lost in ES cells after deletion of intron 2. (A) Genotyping of Ecad-In2flox (+/flox) and Ecad-In2floxdel (+/del) ES cells after transient expression of Cre (left) and offspring of corresponding knock-in mouse strains (right). Primers specific for the floxed (upper panel) and floxdel allele (lower panel) were used. (B) Southern blot analysis of KpnI-fragmented DNA of Ecad-In2flox ES cell clones after Cre expression, using a radioactive probe specific for exon 2. The restriction fragments of the wild-type and Ecad-In2flox alleles migrate at 7.7 kb (black arrow), whereas that for the Ecad-In2floxdel allele migrates at 2.7 kb (white arrow). (C,D) X-gal staining of Ecad-In2flox (C) and Ecad-In2floxdel ES cells (D) shows that expression is lost upon deletion of intron 2 sequences. (E-H) Analysis of differentiated ES cells in teratomas. Expression of ß-gal is seen in teratomas from Ecad-In2flox cells (E), and this is significantly reduced in teratomas without intron 2 (F). (G,H) Sections of teratomas shown in E,F counterstained with Hematoxylin/Eosin. High-level expression in cystic epithelia in Ecad-In2flox (G) is lost in Ecad-In2floxdel teratomas. (I) Semi-quantitative (upper panel) and real-time PCR (lower panel) of both ES-cell lines with primers specific for betageo and Gapd transcripts. A reduction in gene activity is observed in the semi-quantitative and real-time PCR. Values resulting from Gapd real-time PCR were used for standardization. Scale bars: 50 µm in C,D,G,H; 500 µm in E,F.