Chromosomal location of human <i>CEACAM1</i> gene in transgenic mice.

<p><b>A</b>. Human chromosomal spread showing hybridization of human <i>CEACAM1</i> gene probe to chromosome 19 (arrows). <b>B</b>. Murine chromosomal spread of the transgenic mice. <b>C</b>. Hybridization of human <i>CEACAM1</i> gene probe to the murine chromosomal spread, counterstained with DAPI. <b>D</b>. Painting of the murine chromosomal spread from the transgenic mouse. Chromosome 11 is labeled, demonstrating a single integration site on one chromosome (heterozygous).</p

<i>CEACAM1</i> mRNA isoform expression in BAC2-12 transgenic mouse liver, kidney and small intestine.

<p><b>A.</b> RT-PCR analysis for <i>CEACAM1</i> isoforms in transgenic (1–3) or wild type mice (4–6) in liver (1, 4), kidney (2, 5), and small intestine (3, 6). <b>B.</b> RT-PCR analysis for <i>Ceacam1</i> isoforms in transgenic (7–9) or wild type mice (10–12) in liver (7,10), kidney (8,11), and small intestine (9,12).</p

Immunohistochemistry staining of blood neutrophils from BAC2-12 transgenic and wild type mice.

<p><b>A</b>. Transgenic mouse blood neutrophils stained with anti-human CEACAM1 antibody. Mag. 200X (inset photo enlarged 4X). <b>B</b>. Wild type mouse blood neutrophils stained with anti-human CEACAM1. Mag. 200X. <b>C</b>. Transgenic mice blood neutrophils stained with anti-mouse CEACAM1. Mag. 200X (inset photo enlarged 4X). <b>D</b>. Human neutrophils stained with anti-human CEACAM1. Mag. 200X (inset photo enlarged 4X).</p

Transgenic mouse screening.

<p><b>A</b>. PCR screening for <i>CEACAM1</i> expression. Pups 1 to 12 were born after pronuclear microinjection of BAC2. Founders 1, 6 and 12 were positive using <i>CEACAM1</i> primers. <b>B</b>. Western blots of feces using anti-human CEACAM1 mAb demonstrated that BAC2 founders 1, 6 and 12 expressed human CEACAM1 in their gastrointestinal tracts. <b>C</b>. PCR analysis of the F1 generation from founder 12. About 50% of the pups were positive (left side) for human <i>CEACAM1</i> and 100% positive for murine CEACAM1 (right side). <b>D</b>. Western blot analysis of feces using anti-human CEACAM1 mAb. Pups 7, 8, 12, 13, 14, and 18 from founder BAC2-12 were positive. Most fecal samples were collected in the morning (no label) or in the evening (7P, 8P) to determine if time of collection affected the analysis.</p

Expression levels of murine and human CEACAM1 on spleen B and T cells of BAC2-12 transgenic and wild type mice.

<p>Freshly isolated spleen cells (<b>A</b>) and Spleen cells treated with 1 µg/mL anti-CD3 and 2 µg/mL anti-CD28 mAb for three days (<b>B</b>) from TG or WT mice were stained with either the anti-murine CEACAM1-PE or anti-human CEACAM1-PE and co-stained with anti-CD3-FITC and anti-B220-APC. Results shown are representative of three independent experiments with similar results.</p

Confocal analysis of transgenic mouse neutrophils undergoing binding of <i>E. coli</i> expressing recombinant Opa₅₂ protein.

<p><b>Panel 1</b>. <i>E. coli</i> vector control or Opa₅₂ phagocytosed by human neutrophils. <b>A</b>. Vector control <i>E. coli</i> (FITC labeled, green). <b>B</b>. Same as A, stained with anti-human CEACAM1 mAb Alexa 555 labeled (red). <b>C</b>. Same as A, stained with DAPI (blue). <b>D</b>. Overlay of A–C on phase contrast image. Mag 300X. <b>E</b>. Opa₅₂<i>E. coli</i> (FITC labeled, green). <b>F</b>. Same as E, stained with anti-human CEACAM1 Mab Alexa 555 labeled (red). <b>G</b>. Same as E, stained with DAPI (blue). <b>H</b>. Overlay of E–G on phase contrast image. Mag 300X. <b>Panel 2</b>. <i>E. coli</i> vector control or Opa₅₂ phagocytosed by neutrophils from transgenic (A–D) or wild type (E–H) mice. <b>A, E.</b> Vector control <i>E. coli</i> (overlay with FITC, Alexa 555, and DAPI on phase contrast image). <b>B, F.</b> vector control <i>E. coli</i> (FITC labeled, green). <b>C, G.</b> Opa₅₂<i>E. coli</i>. (overlay with FITC, Alexa 555, and DAPI on phase contrast image) <b>D, H.</b> Opa₅₂ expressing <i>E. coli</i> (FITC staining).</p

Immunohistochemistry staining for human and murine CEACAM1 in BAC2-12 transgenic and wild type mice.

<p>Heart (<b>A–C</b>), small intestine (<b>D–F</b>), kidney (<b>G–I</b>), and liver (<b>J–L</b>) in transgenic (<b>A, B, D, E, G, H, J, K</b>) and wild type mice (<b>C, F, I, L</b>) by anti-murine CEACAM1 mAb (CC1) (<b>A, D, G, J, C, F, I, L</b>) or anti-human CEACAM1 mAb (T84.1) (<b>B, E, H, K</b>). Mag. 50X.</p

Immunohistochemistry staining for human and murine CEACAM1 in kidney and liver of BAC2-12 transgenic and wild type mice.

<p><b>A</b>. Wild type mouse kidney. <b>B</b>. Transgenic mouse kidney. <b>C</b>. Transgenic mouse kidney with isotype control antibody. <b>D</b>. Wild type mouse liver. <b>E</b>. Transgenic mouse liver. <b>F</b>. Transgenic mouse liver with isotype control antibody. Note that human CEACAM1 expression is higher than murine CEACAM1 in transgenic mouse kidney. However, in the liver, murine and human CEACAM1 expression in transgenic mice was similar. Bowman's capsule in the kidney is indicated by an arrow (<b>B</b>). Mag. 200X.</p