Volume 122, Issue 3, Pages 689-696 (March 2002) Cdx2 ectopic expression induces gastric intestinal metaplasia in transgenic mice  Debra G. Silberg, Jessica Sullivan, Eugene Kang, Gary P. Swain, Jennifer Moffett, Newman J. Sund, Sara D. Sackett, Klaus H. Kaestner  Gastroenterology  Volume 122, Issue 3, Pages 689-696 (March 2002) DOI: 10.1053/gast.2002.31902 Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 1 Targeting of a 170-kb YAC spanning the mouse Foxa3 (Hnf3γ) gene by insertion of the mouse Cdx2 cDNA. (A) Structure of the unmodified YAC. Restriction sites for NotI (N) are shown. The Foxa3 exons are indicated by the numbered black boxes. The YAC vector arms are depicted as arrowheads (telomers) and open boxes (yeast selectable markers). (B) Two-step procedure for replacement of exon 2 of Foxa3 by the IRES-Cdx2 cassette. The targeting plasmid (circle) consists of the URA3 yeast selectable marker, 2 regions of homology from the Foxa3 gene, and the IRES-Cdx2 cassette. The crossed lines mark the crossover in the first recombination step (Pop-in event). (C) Subsequent counter-selection against the presence of URA3 (Pop-out event) results in the final, modified Foxa3/Cdx2 YAC. Gastroenterology 2002 122, 689-696DOI: (10.1053/gast.2002.31902) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 2 Ectopic expression of Cdx2 in the gastric mucosa by immunohistochemistry and gross appearance of the stomach. Paraffin sections of the stomach from (A) Foxa3/Cdx2 transgenic mice and (B) wild-type littermates were stained with an antibody specific to Cdx2 (1:150). (A) Foxa3/Cdx2 transgenic mice express Cdx2 in a heterogeneous pattern in the nuclei of the epithelium of the gastric glandular mucosa, which is not cell type–specific. (B) The wild-type littermate does not express Cdx2 in the stomach. (C) The Foxa3/Cdx2 transgenic mice develop a small stomach as compared with (D) the wild-type age-matched littermate. Original magnification: A, B, 400×; C, D, line represents 1 cm. Gastroenterology 2002 122, 689-696DOI: (10.1053/gast.2002.31902) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 3 Foxa3/Cdx2 transgenic mice develop intestinal metaplasia with sulfated mucins and alkaline phosphatase expression. Paraffin-embedded sections of the stomach from age-matched (15-month) (B, C, and E) Foxa3/Cdx2 transgenic and (A and D) wild-type littermates were stained with (A–C) alcian blue and (D, E) HID to determine the presence of intestinal-type acidic and sulfated mucins, respectively. The Foxa3/Cdx2 transgenic mice develop intestinal metaplasia with the presence of (B, arrow; C, high power view area indicated in B) alcian blue–positive and (E, arrow) predominately HID-positive sulfated mucins in the distal portion of the stomach. The wild-type gastric mucosa does not contain either (A) alcian blue or (D) HID staining cells. In addition, only the stomachs of (G with higher power inset) Foxa3/Cdx2 transgenic mice express alkaline phosphatase and not their (F) wild-type littermates. Original magnification: A, B, D–G, 200×; C, 600×; G inset, 400×. Gastroenterology 2002 122, 689-696DOI: (10.1053/gast.2002.31902) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 4 Intestine-specific gene expression of the Foxa3/Cdx2 transgenic mice by Northern blot analysis and ribonuclease protection assay. Gene expression was determined on RNA isolated from wild-type (WT) and Foxa3/Cdx2 transgenic mice (TG) glandular stomach or whole stomach and control tissue; K, kidney; L, liver; SI, small intestine; C, colon. Each wild-type RNA sample was directly compared with a transgenic age-matched littermate isolated in the identical manner. Northern analysis of RNA revealed that (A) villin, Fabpi, (B) Gcc and Tff3 are induced in the Foxa3/Cdx2 transgenic mice. Sucrase-isomaltase (sucrase) is expressed only in the small intestine and not in the transgenic mice. (C) Ribonuclease protection assay shows that Muc2 is induced in the stomach of the Foxa3/Cdx2 transgenic mice. GAPDH is a control for RNA loading. Gastroenterology 2002 122, 689-696DOI: (10.1053/gast.2002.31902) Copyright © 2002 American Gastroenterological Association Terms and Conditions