Impaired defense of intestinal mucosa in mice lacking intestinal trefoil factor

Science

Volumn 274, Issue 5285, 1996, Pages 262-265

  Mashimo, Hiroshi ^a   Wu, Deng Chyang ^a   Podolsky, Daniel K ^a   Fishman, Mark C ^a  

^a MASSACHUSETTS GENERAL HOSPITAL   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

COMPLEMENTARY DNA; DEXTRAN SULFATE; INTESTINAL TREFOIL FACTOR; RNA;

ANIMAL CELL; ANIMAL EXPERIMENT; ANIMAL TISSUE; ARTICLE; COLITIS; GENE DISRUPTION; HISTOLOGY; INTESTINE EPITHELIUM; INTESTINE INJURY; INTESTINE MUCOSA; MOUSE; NONHUMAN; NORTHERN BLOTTING; PRIORITY JOURNAL; PROTEIN EXPRESSION; PROTEIN SECRETION; TISSUE REPAIR; WOUND HEALING;

ANIMALS; BASE SEQUENCE; CELL MOVEMENT; COLITIS; COLON; DEXTRAN SULFATE; GENE TARGETING; GROWTH SUBSTANCES; INTESTINAL MUCOSA; MICE; MOLECULAR SEQUENCE DATA; MUCINS; MUSCLE PROTEINS; NEUROPEPTIDES; PEPTIDES; RECOMBINANT PROTEINS;

ANIMALIA; LOTUS;

EID: 0029828745     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.274.5285.262     Document Type: Article

References (34)

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11. A positive-negative selection strategy [S. L. Mansour, K. R. Thomas, M. R. Capecchi, Nature 336, 348 (1988)] was used to enrich for homologous recombination events in the ES cells by selecting neo within the homologous DNA and against a herpes simplex virus thymidine kinase gene (hsv-tk) placed at the end of the targeting vector. Plasmid pPNT [V. L. Tybulewicz, C. E. Crawford, P. K. Jackson, R. T. Bronson, R. C. Mulligan, Cell 65, 1153 (1991)] was used to construct the targeting vector, which was linearized with Not I and electroporated into pluripotent J1 ES cells [E. Li, T. H. Bestor, R. Jaenisch, ibid. 69, 915 (1992)] under conditions previously described [S. M. Strittmatter, C. Fankhauser, P. L. Huang, H. Mashimo, M. C. Fishman, ibid. 80, 445 (1995)]. Disruption of the Itf gene in ES cells after homologous recombination was distinguished from random integration of the targeting vector by Southern blot analysis of genomic DNA from individual clones of cells digested with Xho I. The pITF2 probe identified a 19-kb wild-type fragment and a 12-kb knockout fragment created by introduction of a Xho I site by the homologous insertion of the targeting vector. Approximately 10% of neomycin-resistant ES clones were thus found to have homologous ITF recombination. PCR confirmation of the targeted mutation was carried out with primers spanning exon 2 of ITF (200-base pair product from primer pair sequences 5′-GCAGTGTAACAACCGTGGTTGCT-GC-3′ and 5′-TGACCCTGTGTCATCACCCTGGC-3′) and the neo gene (400-base pair product from primer pair sequences 5′-CGGCTGCTCTGATGCC-GCC-3′ and 5′-GCCGGCCACAGTCGATGAATCC-3′). Tails (∼0.5 cm) from mice were digested in 200 μl of proteinase K (0.5 mg/ml) in 50 mM tris-HCl (pH 8.0) and 0.5% Triton X-100 (Sigma) at 55°C overnight, and 1 μl of this mixture was used directly in a 25-μl reaction volume for PCR (Stratagene). After 10 min of incubation at 96°C, the PCR reaction was hot-started with 72°C hybridization and elongation (60 s) and 96°C denaturation (30 s) for 30 cycles. Portions (10 μl per lane) of the reaction mixture were loaded onto 2% agarose gel. Two independently arising ES clones were used to derive two lines of mice lacking ITF. These mice were screened by Southern genomic blot analysis, as described for ES clones, or by PCR.
12. A positive-negative selection strategy [S. L. Mansour, K. R. Thomas, M. R. Capecchi, Nature 336, 348 (1988)] was used to enrich for homologous recombination events in the ES cells by selecting neo within the homologous DNA and against a herpes simplex virus thymidine kinase gene (hsv-tk) placed at the end of the targeting vector. Plasmid pPNT [V. L. Tybulewicz, C. E. Crawford, P. K. Jackson, R. T. Bronson, R. C. Mulligan, Cell 65, 1153 (1991)] was used to construct the targeting vector, which was linearized with Not I and electroporated into pluripotent J1 ES cells [E. Li, T. H. Bestor, R. Jaenisch, ibid. 69, 915 (1992)] under conditions previously described [S. M. Strittmatter, C. Fankhauser, P. L. Huang, H. Mashimo, M. C. Fishman, ibid. 80, 445 (1995)]. Disruption of the Itf gene in ES cells after homologous recombination was distinguished from random integration of the targeting vector by Southern blot analysis of genomic DNA from individual clones of cells digested with Xho I. The pITF2 probe identified a 19-kb wild-type fragment and a 12-kb knockout fragment created by introduction of a Xho I site by the homologous insertion of the targeting vector. Approximately 10% of neomycin-resistant ES clones were thus found to have homologous ITF recombination. PCR confirmation of the targeted mutation was carried out with primers spanning exon 2 of ITF (200-base pair product from primer pair sequences 5′-GCAGTGTAACAACCGTGGTTGCT-GC-3′ and 5′-TGACCCTGTGTCATCACCCTGGC-3′) and the neo gene (400-base pair product from primer pair sequences 5′-CGGCTGCTCTGATGCC-GCC-3′ and 5′-GCCGGCCACAGTCGATGAATCC-3′). Tails (∼0.5 cm) from mice were digested in 200 μl of proteinase K (0.5 mg/ml) in 50 mM tris-HCl (pH 8.0) and 0.5% Triton X-100 (Sigma) at 55°C overnight, and 1 μl of this mixture was used directly in a 25-μl reaction volume for PCR (Stratagene). After 10 min of incubation at 96°C, the PCR reaction was hot-started with 72°C hybridization and elongation (60 s) and 96°C denaturation (30 s) for 30 cycles. Portions (10 μl per lane) of the reaction mixture were loaded onto 2% agarose gel. Two independently arising ES clones were used to derive two lines of mice lacking ITF. These mice were screened by Southern genomic blot analysis, as described for ES clones, or by PCR.
13. A positive-negative selection strategy [S. L. Mansour, K. R. Thomas, M. R. Capecchi, Nature 336, 348 (1988)] was used to enrich for homologous recombination events in the ES cells by selecting neo within the homologous DNA and against a herpes simplex virus thymidine kinase gene (hsv-tk) placed at the end of the targeting vector. Plasmid pPNT [V. L. Tybulewicz, C. E. Crawford, P. K. Jackson, R. T. Bronson, R. C. Mulligan, Cell 65, 1153 (1991)] was used to construct the targeting vector, which was linearized with Not I and electroporated into pluripotent J1 ES cells [E. Li, T. H. Bestor, R. Jaenisch, ibid. 69, 915 (1992)] under conditions previously described [S. M. Strittmatter, C. Fankhauser, P. L. Huang, H. Mashimo, M. C. Fishman, ibid. 80, 445 (1995)]. Disruption of the Itf gene in ES cells after homologous recombination was distinguished from random integration of the targeting vector by Southern blot analysis of genomic DNA from individual clones of cells digested with Xho I. The pITF2 probe identified a 19-kb wild-type fragment and a 12-kb knockout fragment created by introduction of a Xho I site by the homologous insertion of the targeting vector. Approximately 10% of neomycin-resistant ES clones were thus found to have homologous ITF recombination. PCR confirmation of the targeted mutation was carried out with primers spanning exon 2 of ITF (200-base pair product from primer pair sequences 5′-GCAGTGTAACAACCGTGGTTGCT-GC-3′ and 5′-TGACCCTGTGTCATCACCCTGGC-3′) and the neo gene (400-base pair product from primer pair sequences 5′-CGGCTGCTCTGATGCC-GCC-3′ and 5′-GCCGGCCACAGTCGATGAATCC-3′). Tails (∼0.5 cm) from mice were digested in 200 μl of proteinase K (0.5 mg/ml) in 50 mM tris-HCl (pH 8.0) and 0.5% Triton X-100 (Sigma) at 55°C overnight, and 1 μl of this mixture was used directly in a 25-μl reaction volume for PCR (Stratagene). After 10 min of incubation at 96°C, the PCR reaction was hot-started with 72°C hybridization and elongation (60 s) and 96°C denaturation (30 s) for 30 cycles. Portions (10 μl per lane) of the reaction mixture were loaded onto 2% agarose gel. Two independently arising ES clones were used to derive two lines of mice lacking ITF. These mice were screened by Southern genomic blot analysis, as described for ES clones, or by PCR.
14. A positive-negative selection strategy [S. L. Mansour, K. R. Thomas, M. R. Capecchi, Nature 336, 348 (1988)] was used to enrich for homologous recombination events in the ES cells by selecting neo within the homologous DNA and against a herpes simplex virus thymidine kinase gene (hsv-tk) placed at the end of the targeting vector. Plasmid pPNT [V. L. Tybulewicz, C. E. Crawford, P. K. Jackson, R. T. Bronson, R. C. Mulligan, Cell 65, 1153 (1991)] was used to construct the targeting vector, which was linearized with Not I and electroporated into pluripotent J1 ES cells [E. Li, T. H. Bestor, R. Jaenisch, ibid. 69, 915 (1992)] under conditions previously described [S. M. Strittmatter, C. Fankhauser, P. L. Huang, H. Mashimo, M. C. Fishman, ibid. 80, 445 (1995)]. Disruption of the Itf gene in ES cells after homologous recombination was distinguished from random integration of the targeting vector by Southern blot analysis of genomic DNA from individual clones of cells digested with Xho I. The pITF2 probe identified a 19-kb wild-type fragment and a 12-kb knockout fragment created by introduction of a Xho I site by the homologous insertion of the targeting vector. Approximately 10% of neomycin-resistant ES clones were thus found to have homologous ITF recombination. PCR confirmation of the targeted mutation was carried out with primers spanning exon 2 of ITF (200-base pair product from primer pair sequences 5′-GCAGTGTAACAACCGTGGTTGCT-GC-3′ and 5′-TGACCCTGTGTCATCACCCTGGC-3′) and the neo gene (400-base pair product from primer pair sequences 5′-CGGCTGCTCTGATGCC-GCC-3′ and 5′-GCCGGCCACAGTCGATGAATCC-3′). Tails (∼0.5 cm) from mice were digested in 200 μl of proteinase K (0.5 mg/ml) in 50 mM tris-HCl (pH 8.0) and 0.5% Triton X-100 (Sigma) at 55°C overnight, and 1 μl of this mixture was used directly in a 25-μl reaction volume for PCR (Stratagene). After 10 min of incubation at 96°C, the PCR reaction was hot-started with 72°C hybridization and elongation (60 s) and 96°C denaturation (30 s) for 30 cycles. Portions (10 μl per lane) of the reaction mixture were loaded onto 2% agarose gel. Two independently arising ES clones were used to derive two lines of mice lacking ITF. These mice were screened by Southern genomic blot analysis, as described for ES clones, or by PCR.
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29. Tissues were fixed by perfusion and then immersion in 4% paraformaldehyde (13) and paraffin-embedded sections were stained with polyclonal antibody to ITF or monoclonal antibody to colonic mucin (14). Binding of the primary antibody was visualized by biotinylated secondary antibody, avidin, biotinylated horseradish peroxidase H, and diaminobenzidine tetrahydrochloride reagents per manufacturer's instructions (VectaStain ABC; Vector Laboratories, Burlingame, CA). The slides were counterstained with hematoxylin. Arrows indicate representative goblet cells expressing ITF or mucin (13, 14).
30. Mice received a single intraperitoneal injection of BrdU (50 μg per gram of body weight) from a freshly made stock solution (5 mg/ml) dissolved in phosphate-buffered saline (PBS). The mice were killed 2 hours or 3 days later. At necropsy, a longitudinal 1-cm section of the ascending colon was taken. Samples were immediately placed in a cassette, fixed in Carnoy's fixative overnight, and embedded in paraffin wax. Immunohistochemical detection of BrdU was essentially as described [J. G. Fox et al., Gastroenterology 110, 155 (1996)] using monoclonal antibody to BrdU (Sigma), VectaStain kit, and hematoxylin counterstain. Only crypts longitudinally sectioned and visible in their entire length were analyzed for the number of labeled cells.
31. Mice were given 2.5% (w/v) DSS (molecular weight 40,000; ICN Biomedicals, Aurora, OH) in their drinking water for nine consecutive days and weighed every other day.
32. DSS-treated left colon transections were fixed in 4% paraformaldehyde, mounted in paraffin, and stained with hematoxylin and eosin. Mice were given 2.5% DSS for 9 days.
33. A solution of 4% acetic acid (pH 2.3) was slowly infused (5 μl/mg body weight) 3 cm into the rectal lumen of a lightly anesthetized mouse. After exposure for 30 s, excess fluid was withdrawn and the colon was flushed with 0.3 ml of PBS. After removal of excess fluid, the treatment group was infused (5 μl/mg body weight) with recombinant ITF (1 mg/ml), while the untreated group was infused (5 μl/mg body weight) with bovine serum albumin (BSA; 1 mg/ml). Mice were again infused with ITF (treatment group) or BSA (untreated group) after 12 hours. Mice were killed 30 hours after acid injury and the colons were inspected. Frozen sections of OCT (Miles)-embedded specimen were stained with hematoxylin and eosin.
34. Supported by NIH grants RO1DK46906, P30DK43351, and T32DK07191 and by the Glaxo Institute for Digestive Health (H.M.).