Fig. 1. Expression of COUP-TFII in the stomach using lacZ knock-in model. (A). Generation of the lacZ knock-in allele and generation of floxed COUP-TFII allele. Using homologous recombination, a targeting construct containing nuclear lacZ, Neo/TK and LoxP sites were inserted into the genomic COUP-TFII locus, generating a targeted allele in ES cells. Treatment with Cre recombinase and FIAU selection resulted in a lacZ knock-in allele in which lacZ gene expression was controlled by the endogenous COUP-TFII promoter when recombination took place between the first and the third loxP sites of the targeted allele. In addition, floxed COUP-TFII ES clones that retains COUP-TFII locus but lacks selection markers were generated when recombination took place between the second and the third loxP sites. B, BamHI; H, HindIII; S, SalI; X, XbaI. (B) Cryostat sections of E12.5 heterozygous COUP-TFII/lacZ knock-in embryo were stained (for 2 hours) for lacZ activity. There is relatively high expression in the mesenchymal cells just adjacent to the epithelium. (C). The stomach from a 3-day-old heterozygous knock-in animal was dissected and whole-mount X-gal staining was performed. The boundary between stomach and duodenum is indicated by arrowhead. (D) A cryostat section of stomach from adult heterozygous knock-in animal was stained for lacZ activity (blue) and counterstained with propidium iodide (red). DBA lectin immunostaining denotes the parietal cells (green). There is strong X-gal staining in the base layer and negligible staining in the surface pit layer of the adult Zymogenic unit. m, mesenchyme; e, epithelium; fs, fore-stomach; hs, hind-stomach; d, duodenum; oe, esophagus.