A paradoxical reduction in susceptibility to colonic injury upon targeted transgenic ablation of goblet cells (original) (raw)

Preparation of mITF promoter. To obtain a sufficiently large segment of 5′ flanking promoter region to confer goblet cell–specific expression of the transgene, a 10-kb _Bam_HI DNA fragment including 6,353 bp of 5′ flanking region of mouse ITF (mITF) gene was prepared from a mouse (129/SvJ) genomic bacterial artificial chromosome library (Genome Systems Inc., St. Louis, Missouri, USA) and subcloned into a pBluescript II KS+ phagemid vector (Stratagene, La Jolla, California, USA) (17). Because no appropriate restriction sites were present near the start codon of the mITF gene, a DNA fragment spanning nucleotides –6,353 to +24 of the mITF 5′ flanking region was made by the long and accurate PCR (LA-PCR) technology using LA Taq DNA polymerase (Takara Shuzo Co., Otsu, Japan) and a subcloned 10–kb fragment as a template. Primers used were as follows: T3(+)X: 5′-AGCTTGATAACGCGTTCCTGCAGCCCGGGGA-ATCC-3′ and 5′(–)X: 5′-AGCTTCAGGATCCCTGCACAGGATGTGCAAGGTCA-3′. Both primers contain the _Xho_I restriction site at the 5′ end.

Construction of transgene and generation of β-galactosidase ITF transgenic mice. The β-galactosidase gene was obtained from pSV-β-galactosidase vector (Promega Corp., Madison, Wisconsin, USA) by digestion with _Bam_HI and _Hin_dIII. The resulting 3.7 kb of β-galactosidase gene was inserted into the _Bam_HI and _Hin_dIII sites of pBluescript II KS(+). The 6.3 kb of _Xho_I-digested 5′ end of the mITF promoter was then inserted into _Xho_I sites of pBluescript II KS(+) containing the β-galactosidase gene. The resulting plasmid was digested with _Bam_HI, yielding a 10-kb DNA fragment consisting of the 6.35-kb mITF promoter followed by the β-galactosidase gene (3.7 kb). This fragment was gel purified, dialyzed, and injected into the mouse oocytes. The resulting offspring were screened both by Southern blotting (using the above β-galactosidase fragment as a probe) and by PCR using the following primers directed at the inserted β-galactosidase gene: sense: 5′-CCTGAGGCCGATACTGTCGTC-3′; antisense: 5′-CCAGATAACTGCCGTCACTCC-3′. PCR was performed according to the manufacturer’s guidelines (Promega Corp.) using 0.5 μL of Taq DNA polymerase (Promega Corp.) in a 50-μL reaction containing 1.5 mM MgCl2, 0.2 mM dNTP, 0.8 μM of each primer, and approximately 1 μg of extracted tail DNA. PCR conditions included an initial 10 minutes at 95°C followed by 36 cycles each of 95°C for 1 minute, 58°C for 1 minute, 72°C for 1 minute, and a final 8-minute extension period.

Construction of transgene and generation of mITF/DT-A transgenic mice. The 6.35-kb mITF 5′ flanking sequence digested with _Xho_I (as described above) was subcloned into the _Xho_I site of a pBluescript II KS+ phagemid containing 700 bp of attenuated mutant-type DT-A fragment followed by a polyadenylation signal between _Not_I and _Hin_dIII sites (6–9, 18, 19). This 700-bp DT-A fragment was excised with _Not_I and _Hin_dIII from pGlyCAM/DT-A (G128A) plasmid (6, 7, 20; our unpublished data). The subcloned DNA construct comprising nucleotides –6,353 to +24 of the mITF gene promoter linked to the DT-A fragment was transformed into competent Escherichia coli DH5α cells (CLONTECH Laboratories Inc., Palo Alto, California, USA). The subcloned DNA construct was subjected to restriction mapping and sequencing of the insertion junctions to confirm the correct orientation and preservation of the start codon.

To remove the vector sequence, a 7.1-kb DNA fragment comprising mITF promoter linked to DT-A cDNA was released by digestion with _Bam_HI (mITF promoter 5′ end) and _Not_I (DT-A cDNA 3′ end) using standard techniques. The linearized DNA fragment was purified by agarose gel electrophoresis followed by extraction using QIAquick Gel Extraction Kits (QIAGEN Inc., Santa Clarita, California, USA). After dialysis against injection buffer (5 mM Tris [pH 7.4], 5 mM NaCl, and 0.1 mM EDTA [pH 8.0]), the DNA was used for pronuclear injection of 129/SvJ mouse oocytes that were transferred to pseudopregnant mice using standard techniques (21). Four-week-old live-born mice were screened for the presence of the DT-A transgene by Southern blot analysis using tail DNA. Briefly, after digestion of approximately 1 cm of cut tail with 0.5 mg/mL of proteinase K in digestion buffer (10 mM Tris [pH 7.6], 100 mM NaCl, 10 mM EDTA, and 0.5% SDS) overnight at 55°C, the DNA was purified by conventional phenol/chloroform extraction and ethanol precipitation techniques (22). Subsequently, the DNA was subjected to 1% agarose gel electrophoresis followed by blotting onto nylon membranes (Micron Seperations Inc., Westboro, Massachusetts, USA). The membrane was probed with a 32P-labeled probe — either a 700-bp fragment of DT-A, an 800-bp fragment of 5′-end mITF, or human GAPDH cDNA (CLONTECH Laboratories Inc.) — by random priming, and then exposed at –80°C overnight. Copy number of the transgene was determined by the density ratio of mITF 5′ end to GAPDH in DNA from transgenic mice compared to the ratio of mITF 5′ to GADPH in DT-A–negative littermates.

Ten mice expressing the DT-A transgene were successfully obtained from 24 live-born mice and then bred. All pedigrees were maintained by crosses to nontransgenic 129/SvJ littermates. Mice were kept on a standard chow diet ad libitum in a specific pathogen–free environment.

Histologic examination. Various tissues from β-galactosidase transgene and DT-A transgene–positive and –negative mice were used for histologic examination, immediately after death or sacrifice by cervical dislocation. Samples from stomach, duodenum, jejunum, ileum, and proximal and distal portions of colon were fixed in 10% buffered formalin overnight followed by dehydration in 70% ethanol and embedding in paraffin wax. Serial sections 4 μm thick were deparaffinized, rehydrated, and stained with hematoxylin and eosin for routine histology, or with Alcian blue and periodic acid–Schiff (AB-PAS) to locate goblet cells as described previously (23, 24). The ratio of goblet cells to total epithelial cells was obtained by counting AB-PAS–positive cells and total epithelial cells in 10 high-power fields of well-oriented longitudinal sections under light microscopy. The results are shown as the mean ± SD from 3 mice.

Lac-Z staining. After sacrifice of mice, tissues from β-galactosidase–ITF and control mice were removed and rinsed in cold 1× PBS (pH 7.4) with 2 mM MgCl2. Tissues were fixed for 20 minutes at 23°C in a solution containing 1% formaldehyde, 0.2% glutaraldehyde, 2 mM MgCl2, 5 mM EGTA, and 0.02% NP-40 (Roche Molecular Biochemicals Inc., Indianapolis, Indiana, USA), followed by overnight staining at 37°C in a solution containing 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6, 2 mM MgCl2, 0.1% sodium deoxycholate, 0.02% NP-40, and 1 mg/mL X-galactosidase (5-bromo-4-chloro-3-indolyl-—D-galactoside; Promega Corp.) made up in 1× PBS (free of Mg2 and Ca2). Tissue samples were rinsed in PBS and postfixed in 2% paraformaldehyde with 0.1% glutaraldehyde for 1 hour, then dehydrated in xylene and embedded in paraffin. Sections of 5–10 μm were stained with hematoxylin and eosin Y or eosin Y alone, per standard protocols. (Unless otherwise noted, all reagents were purchased from Sigma-Aldrich, St. Louis, Missouri, USA.)

RNA blot analysis. Eight-week-old mice (both DT-A transgene–positive and –negative) were sacrificed by cervical dislocation. Total cellular RNA from various tissues was extracted with Trizol (Life Technologies Inc., Gaithersburg, Maryland, USA) according to the manufacturer’s protocol. Thirty micrograms each of total RNA from various tissues was denatured with formamide, fractionated by electrophoresis on a 1% formaldehyde agarose gel, and transferred onto nylon membranes (Micron Seperations Inc.). Hybridization was performed under high-stringency conditions using 32P-labeled probes made by the random-primed DNA labeling method. The probes used were DT-A fragment, rITF cDNA (12), PCR products of rpS2 and rSP cDNA (24), and GAPDH (CLONTECH Laboratories Inc.). Rat MUC2 and mouse MUC3 probes corresponding to nucleotide positions 440–657 and 1,062–1,399 respectively were also prepared by PCR (25, 26). The membranes were exposed at –80°C overnight (ITF, pS2, and GAPDH), for 2 days (rSP), or for 3 days (DT-A, MUC2, and MUC3), and signal intensities of trefoil peptides and MUCs were compared with that of GAPDH by scanning densitometry.

Western blotting for ITF and mucin. The entire colon and the distal 6 cm of the small bowel were removed, rinsed in ice-cold 1× PBS, and suspended ice-cold lysis buffer (600 μL/100 mg tissue) containing 1× PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 0.7 mM EDTA, and Roche Molecular Biochemicals complete Mini protease inhibitor cocktail tablets. Samples were homogenized for 30 seconds using a Polytron tissue homogenizer (Brinkmann Instruments Inc., Westbury, New York, USA), and then subjected to centrifugation at 16,000 g for 20 minutes at 4°C. Protein concentration was determined using the DC protein assay kit (Bio-Rad Laboratories Inc., Hercules, California, USA).

Immunoprecipitation was performed on approximately 200 μg of protein using a rabbit polyclonal antibody to ITF peptide (HM 88) (27). Thirty-five microliters of a solution of 20% protein A–Sepharose (Amersham Pharmacia Biotech, Piscataway, New Jersey, USA) was added and the incubation continued for 8 hours at 4°C. After centrifugation at 16,000 g for 2 minutes, the samples were heated in loading buffer for 2 minutes at 85°C, and then loaded onto a 10–20% tricine gel (Novex, San Diego, California, USA) and transferred onto PVDF membrane (Millipore Corp., Bedford, Massachusetts, USA). Blots were blocked for 1 hour at 23°C in a blocking solution containing 5% dry milk, 0.1% BSA, and 0.05% Tween-20 in 1× PBS, and then incubated with the rabbit polyclonal antibody to ITF at 1:500 in the above blocking solution. The blots were then washed in 1× PBS and 0.05% Tween-20 (3 washes of 20 minutes each) and incubated with horseradish peroxidase–linked donkey anti-rabbit antibody (Amersham Life Sciences, Arlington Heights, Illinois, USA) at 1:10,000 in the same blocking solution for 60 minutes at 23°C. Antibody detection was carried out using Renaissance chemiluminescent reagents (NEN Life Science Products Inc., Boston, Massachusetts), according to the manufacturer’s instructions. Western blotting of mouse colonic or small bowel mucin was performed on 60 μg of protein from the above tissue homogenate supernatant. Protein was then separated on a 10–20% tricine gel and transferred and blocked as above. Blots were incubated overnight at 4°C with monoclonal anti-human colonic mucin antibody (R35.2.3) from mice immunized with unfractionated pure colonic mucin (27), followed by incubation with horseradish peroxidase–linked rabbit anti-mouse antibody (Amersham Life Sciences Inc.) at 1:10,000, and antibody detected using the Renaissance chemiluminescent reagents (NEN Life Science Products Inc.) as described above.

DSS and acetic acid–induced experimental colitis. Experimental colitis was induced by two methods using 2 separate standard agents: oral administration of 2.5% (wt/vol) DSS (molecular weight 40 kDa; ICN, Costa Mesa, California, USA) or rectal instillation of 3% acetic acid. For the former treatment, 18 DT-A transgenic mice and 20 normal control mice were treated with 2.5% DSS in their drinking water ad libitum for 12 days (28, 29). Body weight was monitored daily and the survival rate was calculated. Immediately after death or sacrifice at the end of the experiment, tissues were prepared for histologic examination. For the latter treatment, after saline lavage, 3% acetic acid (10 μL/g of body weight) was instilled through a tube inserted 3 cm into the rectum of 28 DT-A transgenic and 28 normal mice under ether anesthesia (23, 24, 30). Survival rate was calculated and mice were examined histologically on days 1, 3, and 5 after administration.

Statistical analysis. Goblet cell number was determined by counting under a microscope. Body weight changes caused by experimental colitis were analyzed using the Student’s t test. A P value of less than 0.05 was considered significant. Survival distribution of mice in experimental colitis was estimated by the Kaplan-Meier method. Difference in survival rate was tested by use of the log-rank test.