Research ArticleExperimental Studies

Organ-specific Responses of Total Body Irradiated Doxycycline-inducible Manganese Superoxide Dismutase Tet/Tet Mice

BYUNG HAN RHIEU, ASHWIN SHINDE, MICHAEL W. EPPERLY, TRACY DIXON, HONG WANG, RICHARD CHAILLET and JOEL S. GREENBERGER
In Vivo November 2014, 28 (6) 1033-1043;

Abstract

Background/Aim: We evaluated doxycycline-inducible manganese superoxide dismutase (MnSODtet/tet) mice after 9.25 Gy total-body irradiation (TBI) or 20 Gy thoracic irradiation. Materials and Methods: Six-week-old MnSODtet/tet or control C57BL/6NHsd mice on or off doxycycline (doxy) in food received 9.25 Gy TBI, were sacrificed at day 19 and bone marrow, brain, esophagus, heart, intestine, kidney, liver, lung, spleen and tongue harvested, total RNAs extracted and transcripts for irradiation response genes quantitated by real time-polymerase chain reaction (RT-PCR). Results: MnSODtet/tet mice only survived with daily injections of doxy beginning 5 days after birth until weaning, at which time they were placed on food containing doxy. Manganese superoxide dismutase (MnSOD) transcript levels were reduced in all tissues except the lung. Adult mice survived with low MnSOD levels, but induced by doxy or TBI. Thoracic-irradiated MnSODtet/tet mice survived past day 120. Conclusion: MnSODtet/tet mice should be valuable for elucidating the role of MnSOD in growth and irradiation response.

Reactive oxygen species (ROS) have been implicated in pathological processes, including diabetes, atherosclerosis, heart failure, neurodegenerative disease, carcinogenesis and chemical- and radiation-induced tissue damage (1-3). Furthermore, the stability of intracellular antioxidant levels has been shown to be critical for embryogenesis, organogenesis, growth and development (4-6).

Ionizing irradiation damage is associated with depletion of cellular antioxidants (7-9) including: glutathione, vitamins such as ascorbate, catalases, glutathione peroxidases and superoxide dismutase. In particular, manganese superoxide dismutase (MnSOD) has been shown to be critical for cell, tissue and organ protection from ionizing irradiation damage (10, 11). Deletion recombinant-negative MnSOD−/− mice are neonatal lethal and die with cardiomyopathy, liver steatosis, neurodegeneration, accumulation of oxidative DNA damage and defects in mitochondrial respiration (6, 12-14). Embryo fibroblast cell lines from MnSOD−/− mice are radiosensitive (15). Tissue specific deletion of the MnSOD gene product using the Cre-loxP system has shown that MnSOD is critical for normal organ development and function (16). Mice with heart-specific or brain-specific MnSOD gene deletion showed progressive dilated cardiomyopathy and spongiform encephalopathy, respectively (16). Transient tissue-specific overexpression of MnSOD has been shown to protect against both acute and late irradiation damage to the lung, esophagus, intestine, urinary bladder and oral cavity in mouse models (17-21).

We recently developed a strain of MnSODtet/tet C57BL/6NHsd mice (22) to study the organ-specific effects of regulated levels of MnSOD on mitochondrial ROS during growth and development. Cell lines from doxycycline (doxy)-inducible MnSODtet/tet mice display MnSOD expression that is regulated by a tetracycline responsive element in the gene promoter. Bone marrow stromal cell lines derived from MnSODtet/tet mice showed that the doxy-induced higher levels of MnSOD correlated with radioresistance, improved cell viability, rapid cell doubling, faster DNA strand-break repair and increased antioxidant stores (22).

In the present study, we quantitated the effects of doxy-induced MnSOD levels in MnSODtet/tet mice on organ-specific gene transcripts in vivo. Doxy administration was required continuously for survival, after birth, and until weaning. MnSODtet/tet adult mice that were taken off doxy to reduce levels of MnSOD, were subjected to total-body irradiation (TBI), sacrificed 19 days later and showed a range of different organ specific RNA transcript responses that were distinct from those of wild-type mice.

Materials and Methods

MnSODtet/tet mice. The generation of MnSODtet/tet mice has been previously described (22). Briefly, to derive MnSODtet/+ mice all pups were genotyped on days 12 to 15 after birth using polymerase chain reaction (PCR) as previously described using the following primers: MnSODwtR 5’ CAT GAT CTG CGG GTT AAT GT 3’, MnSODwtF 5’ AAT TTG GCA CAG GGG AGA C 3’ and MnSODTetR 5’ CAA ATC CTC CTC GTT TTTGG 3’ (22). For the MnSODtet/tet pups to reach weaning, doxy was administered using multiple methods. Initially, breeding pairs were placed on drinking water containing 2.5 mg of doxycycline/ml and water changed weekly. Some of the pups at 5 days after birth had implantation of 21-day release pellets containing 2.5 mg of doxycycline placed subcutaneously. Slow release doxy pellets were used (Innovative Research of America, Sarasota, Florida, USA). For insertion, a small incision was made in the skin and pellets were placed under the skin, skin glued together using opthalmalic glue. To maintain doxycycline levels in breast milk, mothers were injected intraperitonealy (I.P.) with 100 μl containing 2.5 mg doxycycline/ml in water daily. Sub-groups of pups in some experiments beginning on day 5 after birth were injected daily with 50 or 100 μl of water containing 2.5 mg of doxycycline/ml. To increase the number of MnSODtet/tet pups, nursing mothers received food containing doxycycline (Harlan Spraque Dawley, Indianapolis, IN, USA), also supplied to MnSODtet/tet mice after weaning. Food contained 625 mg/kg doxycycline for a daily doxy dose of 1.6 to 2.7 mg of doxy per 3 to 5 gm diet.

Effect of TBI on induction of RNA transcripts in MnSODtet/tet mouse organs. Some six-week-old male and female MnSODtet/tet C57Bl/6 mice continuously receiving doxy in chow were switched to normal chow without doxy and remained off doxy for four days. Mice were then either irradiated to the lethal dose LD50/30 dose of 9.25 Gy TBI or nonirradiated. Subgroups of mice in each group were kept on doxy, and others taken off doxy. Mice were sacrificed at day 19 after TBI and eleven tissues (bone marrow, brain, esophagus, heart, intestine, kidney, liver, lung, muscle, spleen and tongue) were harvested from each mouse for total RNA extraction.

Wild-type (MnSOD+/+) littermate control C57Bl/6 mice underwent a similar protocol. Doxy-treated wild-type mice received doxy in food for two weeks prior to TBI.

RNA was extracted from harvested tissues using the Trizol reagent (Life Technologies, Grand Island, NY, USA) and cDNA synthesized for testing the control (Gusb) for quantitation of levels of expression using primers specific for the promoter regions of: manganese superoxide dismutase (Sod2), glutathione peroxidase 1 (Gpx1), nuclear factor (erythroid-derived2)-like 2 (Nrf2), nuclear factor kappa beta (Nfkβ), transforming growth factor beta (TGFβ), Fas ligand (Fas), transcription factor Sp1 (Sp1), glyceraldehyde-3-phosphate dehydrogenase (Gapdh) and peptidylprolyl isomerase A (Ppia) using RT-PCR as described previously (23). Results were presented as fold increases in RNA above baseline levels of each tissue from either non-irradiated MnSOD+/+ littermate control or MnSODtet/tet mice respectively. Baseline levels allowed determination of the magnitude of detectable elevation in RNA by robot RT-PCR attributable to doxy treatment and/or irradiation in each organ of each mouse genotype.

Thoracic irradiation of MnSODtet/tet mice. MnSODtet/tet or control MnSOD+/+ mice were taken off doxy for five days and then were irradiated to 20 Gy single fraction to thoracic cavity. Some MnSODtet/tet and control were taken off and others maintained on doxy containing food. All thoracic irradiated mice were to be sacrificed at time of moribund condition expected from radiation pulmonary fibrosis in C57BL/6NHsd mice (17).

Statistical methods. For each removed organ (marrow, brain, esophagus, heart, intestine, kidney, liver, muscle, spleen and tongue) and each gene (Sod2, Gpx1, Nrf2, Nfkβ, TGFβ, Fas, Sp1, Gapdh and Ppia), the RNA transcript levels were measured by RT-PCR. These data were normalized by calculating the differences (ΔCt) between the Ct-Gapdh and Ct-Target genes isolated from bone marrow, esophagus, muscle, spleen and tongue. The Ct-Pipa was used to normalize the target genes for RNA isolated form brain, heart, intestine, kidney, liver and lung. The relative increase or decrease in expression was calculated by comparing the reference gene with the target gene (ΔΔCt) and using the formula for relative expression (=2ΔΔCt). Groups were summarized and compared in terms of the relative expression. In each group, these levels of expression were summarized by mean±standard deviation (SD). Expression levels were compared to baseline levels (which were assumed to be constant) with the two-sided one sample t-test. Levels were compared to each mouse group on doxy “for 2 weeks, total body irradiated to 9.25 Gy, then on doxy” to the group “never on doxy, total body irradiated to 9.25 Gy, then remaining off doxy”. Levels were also compared between groups “always on doxy, then total-body irradiated to 9.25 Gy, then remaining on doxy” with groups “non-irradiated always on doxy”. All comparisons were carried out with the two-sided two sample t-test. In all these exploratory studies we did not adjust p-values for multiple tests.

Results

Neonatal lethality of MnSODtet/tet mice in the absence of maternal and newborn doxy administration. MnSODtet/tet mice were bred by mating MnSODtet/+ mice genotyped by RT-PCR using primers specific for the mouse MnSOD gene or MnSODtet transgene using PCR conditions previously published (22). Pups from the first four litters were genotyped at 12 to 15 days after birth and there were no MnSODtet/tet mice identified. The data support previous publications which demonstrated that MnSOD−/− mice died in utero or within five days after birth. Breeding pairs were next placed on water containing 2.5 mg doxycycline (5 mM) to determine if increased levels would activate the MnSODtet/tet gene in pups in utero and during nursing. Six litters were born from females on doxy, but no MnSODtet/tet pups were identified (all were MnSOD+/+ or MnSODtet/+ by genotype) (Table I). In four litters from mothers receiving I.P. doxy, the mothers died of dehydration and produced no tet/tet newborns (Table I). Next, on day 5 after birth, we implanted 21 day release doxy-pellets containing 2.5 mg doxycycline to all newborns in each of 3 litters. Newborns from 3 litters died after implantation of pellets or within 24 h, and no MnSODtet/tet mice survived to time of weaning (Table I).

We next tested intraperitoneal injection of 250 μg of doxycycline into new born pups, but waiting to start until 5 days after birth, then daily until time of weaning, when doxy-containing food was supplied. This method was successful and MnSODtet/tet mice survived to weaning. Most survived to genotyping at days 12 to 15 (Table I).

View this table:
Table I.

Effect of doxy administration to mother and newborns on survival of MnSODtet/tet mice.

Newborn MnSODtet/tet mice were smaller with less than one out of ten surviving to time of weaning (day 21) (Table I, Figure 1). Finally, we optimized the breeding. We decreased doxy levels at injection to 125 μg per day and placed nursing mothers on food containing doxy. There was an increase to 20% in the number of MnSODtet/tet newborns surviving until weaning (Table I). Using these measures, we derived 30 MnSODtet/tet mice surviving to 6-8 weeks of age. Thus, 30 out of 187 genotyped mice were MnSODtet/tet derived from experiments with 128 litters (Table I). The data establish a clear requirement that MnSODtet/tet mice receive doxy during both gestation and the neonatal period.

Surviving MnSODtet/tet mice were smaller at the time of weaning, but once on solid food containing doxy they rapidly gained weight and grew to match the size of littermate MnSOD+/+ and MnSODtet/+ mice (Figure 1). Some MnSODtet/tet mice that survived to reach 6-8 weeks age (adulthood) were taken-off doxycycline. There was an initial weight loss, but after 5 days weight stabilized and mice again appeared healthy and active (Figure 1) despite the absence of doxycycline.

Fertility of MnSODtet/tet mice. We evaluated whether MnSODtet/tet mice were fertile. Two female and two male MnSODtet/tet mice were paired, one male and one female per cage, and allowed to mate. Animals were kept on doxy in the food, as described in the optimal maintenance paradigm in Table I. Both female mice became pregnant and delivered litters. The litters were small containing 2 or 3 pups, which did not survive to time of genotyping. Since, the breeding of heterozygous MnSODtet/+ mice resulted in surviving MnSODtet/tet mice, we no longer attempted to breed MnSODtet/tet mice.

Effect of doxy on levels of gene expression in organs of total-body irradiated wild-type mice. We first examined whether doxy administration altered levels of expression of MnSOD and other irradiation response genes in wild type mice. Wild-type mice showed little effect of doxy on baseline gene expression (Figure 2).

There were no statistically significant differences in levels of Sod2, Gpx1, Nrf2, Nfkβ, TGFβ, Fas and Sp1 in MnSOD+/+ wild-type mice on doxy-treated compared to non-treated wild-type mice.

Figure 1.
Figure 1.

Appearance and growth of MnSODtet/tet mice. A) decreased size and body weight of MnSODtet/tet mouse compared to the MnSOD+/+ mouse at day 14 after birth. B) Change in body weight of MnSODtet/tet mice following 9.25 Gy total body irradiation on Day 0. Mice in the two groups were taken off doxycycline on day 1 and weighed daily. Mice taken off doxycycline-containing food showed a decrease in body weight initially. If the mice were placed back on doxycycline, they returned to normal weight. If the mice were off doxycycline, then loss of weight stabilized after 10 days.

Figure 2.
Figure 2.

Lack of effect of doxycycline on baseline gene transcript expression in MnSOD+/+ wild type mice. The effect of doxycycline on induction of irradiation response genes in MnSOD+/+ wild type mice organs and tissues was determined. Harvested organs and tissues were assayed two weeks after doxycycline treatment by RT-PCR for transcripts Sod2, Gpx1, Nrf2, Nfkβ, TGFβ, Fas and Sp1, as described in the Material and Methods.

Effect of irradiation on levels of gene expression in wild-type mice. Levels of Sod2 and other gene transcripts induced by 9.25 Gy total-body irradiation in wild-type mice were next measured (Figure 3). Previous studies demonstrated that day 19 was the time on the mouse survival curve after the LD 50/30 dose of TBI when death of the 50% destined to die was occurring at a rapid rate. At day 19 after 9.25 Gy TBI, irradiation decreased levels of Sod2 transcripts in brain and kidney of wild-type mice and increased levels in spleen. Doxycycline treatment of irradiated wild-type mice prevented the drop in Sod2 levels in brain (Figure 3A). At this time, levels of Gpx1 were increased in brain and intestine, while decreased in kidney, liver and lung of wild type mice (Figure 3A).

Doxy treated irradiated wild type mice showed decreased Gpx1 in brain and increased Nrf2 in liver, lung and muscle (Figure 3). In contrast, Nfkβ transcripts were decreased in heart, lung and tongue at day 19 after 9.25 Gy TBI of wild-type mice (Figure 3B); TGFβ was increased in brain and liver and decreased in intestine, lung and tongue (Figure 3C). Doxy reduced the irradiation induced TGFβ in brain and increased TGFβ in tongue. Levels of Fas, a marker of apoptosis, were increased in esophagus and tongue (Figure 3C) and decreased in bone marrow. The doxy effect on irradiated wild-type mice was most prominent in the brain. The data support prior studies indicating that doxy protects neurons from ionizing radiation-induced apoptosis (24).

Effect of doxy on gene transcripts in organs of total-body irradiated MnSODtet/tet mice. We first measured baseline transcript levels in MnSODtet/tet mouse organs. Most organs of MnSODtet/tet mice (exception esophagus, intestine and muscle) had lower levels of Sod2 transcripts compared to wild-type despite doxy-containing food (Figure 4A). TBI irradiated MnSODtet/tet mice continuously on doxy showed higher MnSOD levels in bone marrow, lung and intestine. Elevated levels of Sod2 transcripts in intestine of irradiated MnSODtet/tet mice were significantly higher than that in total body irradiated MnSOD+/+ mice (Figure 4A). Despite doxy administration, Sod2 gene transcript levels in brain, heart, liver, spleen and tongue of total-body irradiated MnSODtet/tet mice were lower than in total-body irradiated wild type mice.

MnSODtet/tet mice on doxy had levels of Gpx1 similar to those in wild-type mice (except in bone marrow, kidney and tongue) (Figure 4A). In MnSODtet/tet mice continuously on doxy, irradiation increased Gpx1 levels in intestine but decreased levels in liver and muscle. There were no doxy-specific differences in Gpx1 levels in irradiated MnSODtet/tet mice. Therefore, changes in Gpx1 transcript levels were similar in MnSODtet/tet and wild-type mice.

Baseline RNA transcript levels of Nrf2 in brain, heart, lung and tongue of MnSODtet/tet mice on doxy were lower compared to wild-type mice (Figure 4B). Irradiation increased Nrf2 levels in brain, esophagus and lung of MnSODtet/tet mice on doxy. Irradiated MnSODtet/tet mice continuously on doxy had significantly higher esophagus levels of Nrf2 than MnSODtet/tet mice off doxy (Figure 4B).

Figure 3.
Figure 3.
Figure 3.

Effect of irradiation on induction of gene transcripts in MnSOD+/+ wild type mice. The baseline and radiation-induced changes in expression of irradiation response genes in doxycycline-inducible MnSODtet/tet mice organs and tissues was used to quantitate irradiation effect. Expression levels were standardized to that in non-irradiated MnSOD+/+ wild-type mice. Harvested organs and tissues were assayed 19 days after 9.25 Gy total body irradiation by RT-PCR, as described in the Material and Methods. Results are shown in Panel A: Sod2 and Gpx1; panel B: Nrf2 and Nfkβ; panel C: TGFβ and Fas; and panel D: Sp1. (# represents p-value <0.05 compared to non-irradiated non-doxy baseline wild type, while *shows where the p-value <0.05 compared to MnSOD+/+ mice never on doxy plus 9.25 Gy and remaining OFF doxy).

Nfkβ levels were also lower in brain and tongue but higher in intestine and muscle of MnSODtet/tet mice on doxy compared to wild-type mice (Figure 4B). Irradiation of MnSODtet/tet mice significantly decreased Nfkβ transcript levels in muscle and spleen. There were no differences in levels of Nfkβ between organs and tissues of doxy-treated or untreated irradiated MnSODtet/tet mice.

TGFβ levels were higher in muscle of MnSODtet/tet mice on doxy compared to wild type mice (Figure 4). TBI significantly increased TGFβ in heart of MnSODtet/tet mice on doxy but not in MnSODtet/tet mice off doxy, while levels were higher in the heart than that observed in wild-type mice (Figure 4C).

Fas levels in MnSODtet/tet mice were lower in brain, kidney, lung and tongue when on doxy compared to that in wild-type mice (Figure 4C). Irradiation increased Fas levels in the brain and lung but reduced levels in bone marrow and heart of doxy-treated MnSODtet/tet mice. The bone marrow of irradiated MnSODtet/tet mice on doxy showed a significant decrease in Fas compared to mice off doxy (Figure 4C).

The data establish that MnSODtet/tet mice demonstrate three phases of growth and development: (i) Neonatal lethality in the absence of doxy induction of MnSOD and requirement for doxy by IP injection until weaning, (ii) requirement for doxy in food for survival past weaning and (iii) an adaptation phase at 6-8 weeks of age (adulthood) in which doxy was not required for survival.

Survival of thoracic-irradiated MnSODtet/tet mice. At 200 days all 20-Gy thoracic-irradiated MnSODtet/tet, as well as control MnSOD+/+ mice on or off doxy remained alive and healthy.

Discussion

The absence of MnSOD results in neonatal lethality in mice, characterized by multi-organ dysfunction and pathologic changes associated with oxidative damage (6, 12-14). MnSODtet/tet mice in our study confirmed the neonatal lethality observed in knockout mice, in that absence of doxy induction of MnSOD was associated with in utero or neonatal death. High MnSOD levels were required during gestation and in the neonatal period. MnSOD was clearly required for developing tissues and organs (25). The organs tested in 6-8-week-old MnSODtet/tet mice (except intestine, esophagus and muscle) showed significantly lower baseline levels of MnSOD RNA transcripts compared to wild-type mice. Thus, there were persistent organ-specific differences in MnSOD gene transcript levels in MnSODtet/tet mice that survived on doxy.

Figure 4.
Figure 4.
Figure 4.

Effect of irradiation on gene transcripts in doxycycline-inducible MnSODtet/tet mice. The baseline and radiation-induced changes were determined for levels of expression of irradiation response genes in doxycycline-inducible MnSODtet/tet mice organs. Expression levels were standardized to that in non-irradiated MnSOD+/+ wild-type mice. Harvested organs were assayed 19 days after 9.25 Gy total body irradiation by RT-PCR as described in the Material and Methods. Results are shown in panel A: Sod2 and Gpx1; panel B: Nrf2 and Nfkβ; panel C: TGFβ and Fas; and panel D: Sp1. (The ≠ symbol represents p-value <0.05 compared to baseline non-irradiated non-doxy MnSOD+/+ wild type. The † symbol is the p-value <0.05 compared to Baseline MnSODtet/tet ON doxy. The ♦ symbol has a p-value <0.05 compared to MnSODtet/tet OFF doxy 4 days then irradiated to 9.25 Gy and remaining off doxy. The ○ symbol stands for a p-value <0.05 compared to MnSOD+/+, which were never on doxy, irradiated to 9.25 Gy and then taken off doxy for the remainder of the experiment).

Age-related differences were observed in levels of MnSOD in MnSODtet/tet mice. If MnSODtet/tet mice on doxy survived past weaning, despite constitutively low levels of MnSOD in multiple organs, they continued to survive when taken off doxy. These results establish an adaptation phase in adulthood for survival despite a low level of MnSOD. The data may reflect up-regulation of other antioxidant pathways during growth and maturation. Low, but detectable levels of MnSOD have been reported in bone marrow stromal cells derived from MnSODtet/tet mice (22). The low basal level of MnSOD was enough to allow survival of 6-8 week old MnSODtet/tet mice even without doxy and for 19 days after 9.25 Gy TBI. Other tet-based inducible transgenic models have also shown the phenomenon of transgene leak in vitro and in vivo (26-33). Mechanisms such as protein stabilization may have contributed to survival of MnSODtet/tet mice during adulthood. We also cannot rule out the possibility that MnSODtet/tet mice may have displayed greater rates of death after 19 days.

MnSODtet/tet mice had significantly lower body weight compared to wild type mice during the neonatal period. MnSOD levels correlated with weight of MnSODtet/tet mice. MnSOD is known to be required for signal transduction pathways involved in cell proliferation and growth, including the growth stimulatory function of the mTOR signaling and growth inhibitory function of GSK-3β signaling (34). If doxy was discontinued in neonatal mice, we observed an immediate weight reduction. Skeletal muscle-specific MnSOD-knockout mice have exercise intolerance and a more rapid fatigue (35). The immediate weight loss after cessation of doxy may have been from reduced mobility, increased fatigability and reduced food intake in neonatal MnSODtet/tet mice.

Doxycycline modulated the irradiation response in MnSODtet/tet mice most prominently in the brain. The brain of wild-type C57BL/6NHsd mice that received doxy had radiation-induced gene expression changes that reflected radioprotection, including: higher levels of MnSOD, lower TGFβ and lower Fas. The tetracycline family of antibiotics has been shown to display neuroprotective effects in hypoxia-ischemia animal models and protect against apoptosis from oxidative stress or inflammation (36, 37). Tetracycline is also known to be a radioprotector that inhibits irradiation-induced DNA fragmentation protecting neurons from apoptosis (24).

MnSODtet/tet mice on doxy had MnSOD levels that were still significantly lower than those in wild-type mice, and transgene expression varied between organs. The intestine had high levels of MnSOD, perhaps explained by the fact that the luminal epithelium of intestine is situated at the initial interface between the absorptive cellular environment and the highly concentrated doxycycline in the food given to the mice. Therefore, the inducible gene expression in intestine may have been higher. TBI significantly induced MnSOD transcripts in the lungs of MnSODtet/tet mice on doxy, to levels higher than that in lungs of mice off doxy. That the lungs of 20 Gy thoracic irradiated MnSODtet/tet mice either on or off doxy at day 120 showed no clinical signs of irradiation pulmonary fibrosis is consistent with the TBI data showing high MnSOD levels.

MnSODtet/tet mice revealed several limitations as a model system. It was difficult to obtain enough viable mice for large experiments given their extreme fragility during the neonatal period. The present gene expression data in TBI mice were limited to a single 19 day time point. Expression patterns of radiation response genes at earlier or later time points may be relevant and are being evaluated in new experiments. The present MnSODtet/tet total body inducible system makes difficult any study of the effects of a change on one specific organ. Other organ-specific transgenic approaches used a Tet-inducible system and Cre/lox P system, which may be of greater value to address organ-specific MnSOD levels, as well as effects on survival of mice during early development (38-41).

The high level of regulatable MnSOD in the intestine of our MnSODtet/tet mice may be ideal for studying the role of MnSOD in the intestinal radiation injury model. Thus, MnSODtet/tet mice should be valuable for understanding the role of MnSOD levels in organ function during gestation in utero, growth and development, as well as response to ionizing irradiation.

Acknowledgements

Supported by U19A168021 Grant from the NIAID/NIH. This project used the UPCI animal facility that is supported in part by award P30CA047904.

  • Received June 11, 2014.
  • Revision received October 15, 2014.
  • Accepted October 17, 2014.

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Organ-specific Responses of Total Body Irradiated Doxycycline-inducible Manganese Superoxide Dismutase Tet/Tet Mice
BYUNG HAN RHIEU, ASHWIN SHINDE, MICHAEL W. EPPERLY, TRACY DIXON, HONG WANG, RICHARD CHAILLET, JOEL S. GREENBERGER
In Vivo Nov 2014, 28 (6) 1033-1043;
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