Genotyping.

Tissue was lysed with 25 mM sodium hydroxide (pH 12.0) at 95°C for 25 min. Then the solution was neutralized with the same volume of 40 mM Tris buffer (pH 5.0) as the volume of sodium hydroxide. This solution was used for PCR. For Cre and BmpRIa, PCR was done with RedTaq (Sigma), and KlenTaq (DNA Polymerase Facility, Washington University, St. Louis, MO) was used for LacZ PCR. Primers were as follows: Cre forward AGG GAT CGC CAG GCG TTT TC and reverse GTT TTC TTT TCG GAT CCG CC, BmprIa forward GCA GCT GCT GCT GCA GCC TCC and reverse TGG CTA CAA TTT GTC TCA TGC, LacZ forward GTT GCA GTG CAC GGC AGA TAC ACT TGC TGA and reverse GCC ACT GGT GTG GGC CAT AAT TCA ATT CGC. For genotyping from paraffin-embedded tissue slides, EX-WAX DNA Extraction Kit (Chemicon International, Temecula, CA) was used to extract genomic DNA.

Immunofluorescence.

Stomachs were prepared and stained by methods modified from Ramsey et al. (31). Briefly, stomachs were inflated with freshly prepared 10% formalin fixative and suspended in fixative for 4 h at room temperature, followed by multiple rinses in 70% EtOH, arrangement in 2% agar in a tissue cassette, and routine paraffin processing. Sections (5 μm) were deparaffinized and rehydrated, and antigen-retrieval was performed by boiling in 10 mM sodium citrate pH 6.0. Slides were blocked in 1% BSA, 0.3% Triton X-100 in PBS, then incubated in primary followed by secondary antibodies (see below) and/or with the fluorescently labeled lectin Griffonia simplicifolia-II (GS-II, neck cells; 1:1,000, Invitrogen, Carlsbad, CA). Slides were finally incubated 5 min in 1 μg/ml bisbenzimide (Invitrogen) before being mounted in 1:1 glycerol:PBS. Fluorescence microscopy and imaging were performed as described (31).

The following primary antibodies were used for immunostaining in this study: rabbit (1:10,000), goat (1:2,000) anti-human gastric intrinsic factor (gifts of Dr. David Alpers, Washington University), rabbit anti-H⁺/K⁺ ATPase α-subunit [1:1,000, gift of Dr. Michael Caplan (5)], rabbit anti-γH2AX (1:50, Cell Signaling Technology, Danvers, MA), goat anti-bromodeoxyuridine (BrdU) (1:2,000, gift of Jeffrey Gordon, Washington University), and rabbit anti-Cre (1:1,000, Novagen, San Diego, CA). Secondary antibodies used were AlexaFluor (488, 594, or 647)-conjugated donkey anti-goat or anti-rabbit (1:500, Invitrogen).

LacZ staining.

LacZ staining was modified from a described method (17). Tissue was fixed in LacZ fix for 4 h at 4°C and washed in LacZ wash buffer three times. Tissue was equilibrated in 30% sucrose-PBS overnight at 4°C, was embedded in OCT (Sakura, Torrance, CA), and was frozen in dry ice. The frozen block was cryosectioned at 14-μm thickness. The section was fixed again for 10 min in LacZ fix and washed in LacZ wash buffer three times. Then the section was incubated in LacZ stain 6 h at 30°C and washed in PBS three times. The section was postfixed in LacZ fix at room temperature for 10 min, dehydrated through ethanol and xylene, and then mounted in Cytoseal XYL (Richard-Allan Scientific, Kalamazoo, MI).

BrdU labeling.

We injected 15 μl of BrdU solution (8 mg/ml bromodeoxyuridine, 0.8 mg/ml fluorodeoxyuridine, Sigma) per 1 g body wt intraperitoneally 90 min before euthanasia. Staining was done as described above.

TUNEL staining.

Stomachs were inflated with freshly prepared formalin and suspended in fixative overnight at 4°C, followed by multiple rinses in 70% EtOH, arrangement in 2% agar in a tissue cassette, and routine paraffin processing. Sections (5 μm) were deparaffinized and rehydrated, then treated with proteinase K (20 μg/ml) at room temperature for 15 min. Tissue sections were incubated with terminal transferase (TdT) and biotin-dUTP (Roche) at 37°C for 1 h, followed by AlexaFluor 488 conjugated streptavidin (1:500, Invitrogen) and Lectin GS-II, AlexaFluor 647 Conjugate (1:1,000, Invitrogen), incubation at room temperature for 1 h.

Western blot.

For Western blot analysis, stomach tissue was frozen in liquid nitrogen and ground with mortar and pestle. Ground powder was lysed in 8 M urea buffer (8 M urea, 0.19 M Tris·HCl pH 6.8, 1% SDS). Proteins were separated on a NuPAGE4–12% polyacrylamide gel (Invitrogen) and transferred to polyvinylidene difluoride membrane. Primary antibodies used for blotting were rabbit anti-γH2AX (1:1,000, Cell Signaling Technology) and rabbit anti-α-tubulin (1:1,000, Cell Signaling Technology). Secondary antibody was horseradish peroxidase (HRP)-conjugated donkey anti-rabbit IgG (1:2,000, Jackson ImmunoResearch, West Grove, PA). Immobilon Western Chemiluminescent HRP Substrate (Millipore) was used for detection.

The gastric phenotype is caused by Cre genotoxicity.

Cre recombinase can cause DNA damage which leads to DNA repair, cell cycle inhibition, and/or cellular apoptosis. Consistent with genotoxicity as a mechanism for the gastric phenotype following induction of Cre recombinase, we observed an increase in apoptosis in CAGGCreER^TM stomachs after tamoxifen injection compared with controls (Fig. 3A). Also, the DNA double-strand break marker, γH2AX, was positive only in CAGGCreER^TM mouse stomachs after tamoxifen injection (Fig. 3B). This was confirmed with Western blot (Fig. 3C). Finally, we assessed levels of several DNA damage-responsive transcripts: p53, Ddit3, and Gadd45a expression levels were statistically significantly (P < 0.05 to <0.01) increased in CAGGCreER^TM stomachs after tamoxifen injection compared with controls (Fig. 3D).

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Fig. 3.

Genotoxicity induced by Cre recombinase. A: TdT-mediated dUTP nick-end labeling (TUNEL; green) labeling of wild-type mice 7 days (left) as well as CAGGCreER^TM mice 3 days (middle), and 7 days following tamoxifen injection. Neck and/or SPEM cells are labeled with GS-II (purple). Note abundant TUNEL-positive nuclei (e.g., arrowheads) indicating diffuse cell death in mice with Cre recombinase activated. B: CAGGCreER^TM mice 3 days following injection of vehicle alone (left) and tamoxifen (right). Note increased genotoxicity in Cre recombinase activated, tamoxifen-treated mice with increased nuclei positive for γH2AX (purple; e.g., green arrowheads), indicating DNA damage. GS-II is green, GIF is red. C: Western blot showing increase in γH2AX in 3 days following tamoxifen treatment in 3 different CAGGCreER^TM mice relative to 2 different control mice. Anti-α-tubulin of same lanes was used as loading control. D: abundance in whole stomach of DNA damage transcripts measured by quantitative RT-PCR (qRT-PCR) for each of the indicated genes is expressed as a ratio of 3 days following tamoxifen-treated CAGGCreER^TM mice relative to control. Note in all figures: qRT-PCR expressed as Log₂ scale. *P < 0.05; **P < 0.01.

Cre recombinase induces genotoxicity specifically in the stomach and not other organs.

Because CreER^TM expression in our experiments was driven by a β-actin promoter with relatively universal expression, all cells could theoretically be affected by Cre recombinase following tamoxifen treatment. We tested other organs (small intestine, large intestine, liver, pancreas, spleen, urinary bladder, lung, and skin) for Cre induced changes. All other organs in tamoxifen-treated CAGGCreER^TM mice were indistinguishable from controls [Fig. 4, A–D; n = 31 mice in 5 experiments; see also Mysorekar et al. (23) for specific analysis of bladder] with the exception of small intestine, which showed mild, focal crypt hyperplasia with an increase in crypt apoptotic figures (Fig. 4A). We assessed levels of DNA-damage-responsive transcripts by qRT-PCR and also found no difference between tamoxifen-treated CAGGCreER^TM mice and tamoxifen-treated wild-type controls with the exception of liver, which showed increase in p53, Ddit3, and Gadd45a expression (data not shown).

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Fig. 4.

Other organs are not affected by Cre as severely as stomach. Small intestine (A), large intestine (B), liver (C), and pancreas (D) from vehicle-treated (top) and tamoxifen-treated (bottom) CAGGCreER^TM mice 7 days following injection. Note that organs with activated Cre recombinase do not differ substantially from control, although there is mild crypt hyperplasia in small intestine with an increase in apoptotic (e.g., orange arrowhead) and mitotic (e.g., white arrowhead) figures (higher magnification panel at bottom left). E: constitutive (i.e., prior to tamoxifen treatment) Cre expression levels in various organs in CAGGCreER^TM mice using qRT-PCR. Stomach has the highest Cre recombinase expression level except liver (n = 3 animals/group; P < 0.07 for stomach vs. small intestine). *P < 0.05; ***P < 0.001.

Cre genotoxicity positively correlates with the Cre transgene expression level.

We next performed experiments to test whether stomach specificity of the genotoxicity induced by CAGGCreER^TM was due to higher levels of Cre transgene expression in the stomach relative to other organs. qRT-PCR showed that, in CAGGCreER^TM mice, stomach had the second highest constitutive levels of Cre transgene expression of the organs examined (Fig. 4, C and E). Cre expression in small intestine was twofold less than it was in stomach (although the difference was not technically statistically significant, P < 0.07 by one-way ANOVA followed by either Dunnett or Tukey post hoc multiple comparison correction; n = 3 mice/group), and small intestine had minimal histological phenotype and no increase in DNA damage marker (Fig. 4, A and E). Large intestine and pancreas, neither of which had shown histological phenotype nor DNA damage marker increase, had fourfold less Cre expression than stomach (P < 0.001 for large intestine and P < 0.001 for pancreas). The exception to the trend was liver, which had statistically higher expression (P < 0.05) than stomach but did not show histological damage, although liver had shown increased DNA damage markers. In sum, the data show a trend for higher Cre transgene expression to correlate with increased induction of DNA damage and/or histological injury following tamoxifen injection.

To further explore this interpretation, we analyzed tamoxifen-inducible Cre activity in mice expressing Cre under a different global promoter. R26CreERT mice, which also have broad Cre expression, were analyzed at the same tamoxifen dose and schedule. The Cre genotoxic phenotype was not observed in R26CreERT mouse stomach after tamoxifen injection (Fig. 5A). As would be predicted if genotoxicity correlated positively with level of Cre expression, the constitutive gastric Cre transgene expression level was 2.6-fold less in R26CreERT vs. CAGGCreER^TM mice (P < 0.001; n = 3 mice/group). Thus, overall, the Cre genotoxic phenotype correlated with Cre transgene levels both across organs and in the same organ using promoters driving different constitutive levels (see also discussion for why liver might be a partial exception to this pattern).

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Fig. 5.

R26CreER^T mice have lower gastric Cre recombinase expression than CAGGCreER^TM mice and do not show Cre genotoxicity. R26CreERT mice do not show the Cre genotoxic phenotype induced in CAGGCreER^TM mice following the same dose and schedule of intraperitoneal tamoxifen injection (5 mg/20 g for 3 days; cf. Fig. 1D). B: R26CreER^T stomachs have 2.6-fold lower constitutive Cre recombinase expression level than CAGGCreER^TM stomachs. ***P < 0.001.

Stomach has regenerative capacity to recover from Cre genotoxicity-induced injury.

Given the extent of the Cre-induced damage to the gastric mucosa, we expected that animals would either not survive long term because of stomach erosion and hemorrhage or that they would develop tumors arising from the regenerative foci. Surprisingly, however, of the seven tamoxifen-treated CAGGCreER^TM mice we have followed longer than 2 wk after Cre induction, three survived until we euthanized them several months later. Figure 6 shows the gastric mucosa of such survivors. One animal (taken at 11 wk) showed near complete recovery of the entire mucosa. No hyperplastic regions remained, and nearly all gastric units showed parietal cells with more or less normal zymogenic cell differentiation (Fig. 6A, left). There was some residual unevenness of the luminal surface, and many regions exhibited some degree of irregularity in organization among neighboring gastric units (see Fig. 6B, left, where units normally organized like test tubes in a rack show unit lumens that are not perpendicular to the muscle layers and are of varying lengths in the muscle to gastric surface dimension). The abundant stroma of earlier time points had regressed, as had the inflammation. In another long-term animal, parietal cells and zymogenic cells had returned diffusely, but there was considerably more irregularity in gastric unit length and organization with obvious hyperplasia still present in some regions (Fig. 6, A and B, right). BrdU labeling showed that proliferation rates matched histology. In regions that appeared normal, BrdU incorporation had returned to normal rates; in regions that were histologically hyperplastic and irregular, BrdU immunoreactivity indicated proliferation was still increased (Fig. 6C). Overall, the results suggest that, at least in some mice, the severe mucosal injury induced by Cre recombinase can be repaired, possibly by proliferation and subsequent differentiation of stem cells located in the hyperplastic foci observed at 14 days (e.g., Figs. 1, D and E, and ​and2C2C).

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Fig. 6.

Mice 11–12 wk after injury show substantial regeneration of mucosa. A–C: CAGGCreER^TM mice 11 (left) and 12 (right) wk following tamoxifen injection. Regions with more (A) and less (B) pronounced recovery from Cre recombinase damage are depicted. C: gastric units showing proliferation (anti-BrdU, purple) with lumen to left, base to right; neck cells (green, GS-II) and zymogenic cells (red, GIF) are also depicted. Left: in well-recovered regions, both normal, non-SPEM differentiation of neck and zymogenic cells can be seen, corresponding with normal rates of proliferation. Right: in regions where neck and zymogenic cell architecture is still metaplastic (note overlap of GS-II and GIF labeling), proliferation is still increased.

Tamoxifen dosage and frequency can be titrated to achieve loxP-specific recombination without nonspecific genotoxicity.

Tamoxifen induces Cre recombinase activity, because CreER^TM resides in the cytoplasm unless it encounters tamoxifen, which mediates Cre translocation into the nucleus where it can bind genomic DNA. (9). A loxP-flanked gene is permanently deleted in a given cell, as soon as sufficient Cre recombinase activity is present to bind, loop out, and recombine the sequence flanked by loxP sites. After loxP-flanked gene excision or in the absence of any true loxP sites, it follows that increasing Cre concentration in the nucleus would serve only to increase the potential for nonspecific recombination. Indeed, in vitro, Cre activity toward non-loxP sites has been shown to be dose dependent (1, 18). Since tamoxifen-inducible Cre recombinase activity is a critical tool for examination of gene function in organs like the stomach, which exhibits constant homeostatic turnover and largely assumes its adult differentiation state well after birth, it was important to test whether a dose of tamoxifen could be achieved that would not induce nonspecific genotoxicity. To verify the efficiency of DNA recombination in the stomach, we crossed CAGGCreER^TM mice with R26R reporter mice, where lacZ gene expression is driven by a Rosa26 promoter followed by floxed STOP cassettes. With a dose of 5 mg of tamoxifen per 20 g body wt and 3 days of daily injection (the dose used that generated the phenotypes examined above), DNA recombination efficiency was 100% in the stomach epithelium, both in corpus and antrum, within 3 days (Fig. 7A) To determine whether we could lower the dose of tamoxifen without losing efficiency of loxP-flanked gene excision, we lowered tamoxifen dosage to 1 mg per 20 g body wt. At this dose, we did not observe any Cre genotoxic effect in the stomach. However, DNA recombination efficiency was low. Surface pit cells and zymogenic cells showed almost 100% recombination efficiency, however, the efficiency in parietal cells and neck progenitor cells was less than 5% (Fig. 7B), so we increased frequency, maintaining dosage at 1 mg. When we injected tamoxifen for 7 consecutive days, the recombination efficiency was again 100% (Fig. 7D); however, the stomach showed focal metaplasia on day 7, although other animals in this cohort recovered by day 14 (not shown). We next decreased tamoxifen dosage to 0.75 mg per 20 g body wt with the same frequency. At this schedule, recombination in all epithelial cell types except parietal cells was near 100% and nearly 100% of gastric units showed this pattern (Fig. 7E). With the same dosage and schedule, DNA recombination efficiency was ~90% in antrum where parietal cells are not present (data not shown). In corpus, the LacZ histochemical staining pattern coincided with Cre immunofluorescence staining (Fig. 8). With 5 mg tamoxifen injection per 20 g body wt for 3 days, almost every cell expressed nuclear Cre (Fig. 8A), whereas only the zymogenic cell lineage had nuclear Cre staining with 0.75 mg tamoxifen injection per 20 g body wt for 7 days (Fig. 8D). Wild-type mouse stomach showed only background staining (Fig. 8B), and CAGGCreER^TM mouse stomach with vehicle injection showed cytoplasmic Cre staining with occasional weak nuclear staining in zymogenic cells. These data show that it is possible to obtain specific recombination at floxed alleles without Cre genotoxicity in tamoxifen-inducible Cre lines by titrating tamoxifen dosage and frequency.

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Fig. 7.

Nongenotoxic tamoxifen dosing induces specific Cre recombination. Gastric units with lumens to left are depicted from CAGGCreER^TM mice crossed on an R26R reporter background. Recombination efficiency is evidenced by lacZ-positive cells (blue) and nuclear fast red counterstain. Various doses of tamoxifen and injection frequencies (“days” indicate consecutive days of injection) are shown. Mice were euthanized on the 7^th day following the first tamoxifen injection. Note higher doses give higher recombination efficiency but also cause metaplasia as already shown (see, e.g., A). All micrographs at same magnification, scale bar in A.

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Fig. 8.

Cre recombinase immunostaining confirms lacZ staining. A: CAGGCreER^TM mice treated with 5 mg tamoxifen for 3 days. Note nuclear stain in nearly all epithelial cells (e.g., arrowheads; cf. Fig. 7A for the same conditions using X-gal). B: wild-type mice treated with 0.75 mg tamoxifen for 7 days (note only background staining). C: CAGGCreER^TM mice treated with vehicle for 7 days (note almost all cells have diffuse cytoplasmic Cre, although occasional cells show some nuclear labeling, arrowhead). D: CAGGCreER^TM mice treated with 0.75 mg tamoxifen for 7 days. Most zymogenic lineage cells have nuclear Cre staining in CAGGCreER^TM mice treated with 0.75 mg tamoxifen for 7 days whereas parietal cells do not (arrowheads show zymogenic cell nuclear Cre; cf. LacZ staining in Fig. 7E).