Identification of gene mutations associated with the phenotype of short-limb mice emerging from a foundation colony of severely immunodeficient mice.

Here, we report the identification of causative genes for limb-shortening in individuals repeatedly found in a population of severely immunodeficient NOG mice maintained via sibling mating. First, we conducted a pedigree survey to determine whether limb-shortening was a recessive genetic trait and then identified it using a crossing test. Simultaneously, the symptoms were identified in detail using pathological analysis. Accordingly, a mouse strain exhibiting a recessive trait caused by a single gene trait and similar symptoms was identified, suggesting growth differentiation factor 5 (Gdf5) as a causative gene. Genome walking via PCR and sequence analysis of Gdf5 revealed a deletion of approximately 1.1 kb from the latter half of exon 2 of Gdf5. Furthermore, we established NOG-Gdf5bpJic by removing other modified genes and confirmed that the inheritance pattern was reconfirmed semi-dominant. In recent years, regenerative medicine research using immunodeficient mice has been actively conducted, and this murine strain is expected to contribute to niche stem cell analysis and transplantation research.

Improvement of multilineage hematopoiesis in hematopoietic stem cell-transferred c-kit mutant NOG-EXL humanized mice.

Human hematopoietic stem cell (HSC)-transferred humanized mice are valuable models for exploring human hematology and immunology. However, sufficient recapitulation of human hematopoiesis in mice requires large quantities of enriched human CD34+ HSCs and total-body irradiation for adequate engraftment. Recently, we generated a NOG mouse strain with a point mutation in the c-kit tyrosine kinase domain (W41 mutant; NOGW mice). In this study, we examined the ability of NOGW mice to reconstitute human hematopoietic cells. Irradiated NOGW mice exhibited high engraftment levels of human CD45+ cells in the peripheral blood, even when only 5,000-10,000 CD34+ HSCs were transferred. Efficient engraftment of human CD45+ cells was also observed in non-irradiated NOGW mice transferred with 20,000-40,000 HSCs. The bone marrow (BM) of NOGW mice exhibited significantly more engrafted human HSCs or progenitor cells (CD34+CD38- or CD34+CD38+ cells) than the BM of NOG mice. Furthermore, we generated a human cytokine (interleukin-3 and granulocyte-macrophage colony-stimulating factor) transgenic NOG-W41 (NOGW-EXL) mouse to achieve multilineage reconstitution with sufficient engraftment of human hematopoietic cells. Non-irradiated NOGW-EXL mice showed significantly higher engraftment levels of human CD45+ and myeloid lineage cells, particularly granulocytes and platelets/megakaryocytes, than non-irradiated NOGW or irradiated NOG-EXL mice after human CD34+ cell transplantation. Serial BM transplantation experiments revealed that NOGW mice exhibited the highest potential for long-term HSC compared with other strains. Consequently, c-kit mutant NOGW-EXL humanized mice represent an advanced model for HSC-transferred humanized mice and hold promise for widespread applications owing to their high versatility.

Establishment of a human microbiome- and immune system-reconstituted dual-humanized mouse model.

Humanized mice are widely used to study the human immune system in vivo and investigate therapeutic targets for various human diseases. Immunodeficient NOD/Shi-scid-IL2rγnull (NOG) mice transferred with human hematopoietic stem cells are a useful model for studying human immune systems and analyzing engrafted human immune cells. The gut microbiota plays a significant role in the development and function of immune cells and the maintenance of immune homeostasis; however, there is currently no available animal model that has been reconstituted with human gut microbiota and immune systems in vivo. In this study, we established a new model of CD34+ cell-transferred humanized germ-free NOG mice using an aseptic method. Flow cytometric analysis revealed that the germ-free humanized mice exhibited a lower level of human CD3+ T cells than the SPF humanized mice. Additionally, we found that the human CD3+ T cells slightly increased after transplanting human gut microbiota into the germ-free humanized mice, suggesting that the human microbiota supports T cell proliferation or maintenance in humanized mice colonized by the gut microbiota. Consequently, the dual-humanized mice may be useful for investigating the physiological role of the gut microbiota in human immunity in vivo and for application as a new humanized mouse model in cancer immunology.

A case of severe paraquat poisoning treated by continuous hemodiafiltration without sequelae.

Background: In a suicide attempt, a 49-year-old male ingested pesticides. He arrived at the hospital restless and vomiting blue liquid. Case Presentation: The patient was diagnosed with paraquat poisoning at a lethal dose and experienced renal dysfunction during treatment. He underwent continuous hemodiafiltration (CHDF). Hemodialysis was temporarily initiated and found to improve renal function. He was discharged on day 36 in good condition. He remains well with only mild renal impairment and no pulmonary fibrosis, 240 days after the incident. The fatality rate of paraquat poisoning is approximately 80%, regardless of the treatment. Early hemodialysis combined with CHDF within 4 h has been reported to be effective. In this case, CHDF was initiated approximately 3 h after paraquat administration and showed a successful outcome. Conclusion: CHDF should be performed as soon as possible to treat paraquat poisoning.

Efficient differentiation of human neutrophils with recapitulation of emergency granulopoiesis in human G-CSF knockin humanized mice.

Neutrophils are critical mediators during the early stages of innate inflammation in response to bacterial or fungal infections. A human hematopoietic system reconstituted in humanized mice aids in the study of human hematology and immunology. However, the poor development of human neutrophils is a well-known limitation of humanized mice. Here, we generate a human granulocyte colony-stimulating factor (hG-CSF) knockin (KI) NOD/Shi-scid-IL2rgnull (NOG) mouse in which hG-CSF is systemically expressed while the mouse G-CSF receptor is disrupted. These mice generate high numbers of mature human neutrophils, which can be readily mobilized into the periphery, compared with conventional NOG mice. Moreover, these neutrophils exhibit infection-mediated emergency granulopoiesis and are capable of efficient phagocytosis and reactive oxygen species production. Thus, hG-CSF KI mice provide a useful model for studying the development of human neutrophils, emergency granulopoiesis, and a potential therapeutic model for sepsis.

Pathogenesis of murine astrovirus in experimentally infected mice.

Astroviruses are often associated with gastrointestinal diseases in mammals and birds. Murine astrovirus (MuAstV) is frequently detected in laboratory mice. Previous studies on MuAstV in mice did not report any symptoms or lesions. However, little information is available regarding its pathogenicity in immunodeficient mice. Therefore, in this study, we experimentally infected germ-free NOD.Cg-PrkdcscidIl2rgtm1Sug/ShiJic (NOG) mice, which are severely immunodeficient, with MuAstV. Germ-free mice were used for experimental infection to eliminate the effects of intestinal bacteria. Mice in each group were then necropsied and subjected to PCR for MuAstV detection, MuAstV RNA quantification in each organ, and histopathological examination at 4 and 28 days post inoculation (DPI). Tissue samples from the small intestine were examined by transmission electron microscopy. No symptoms or abnormalities were detected in any mice during necropsy. The MuAstV concentration was highest in the lower small intestine, where it increased approximately 8-fold from 4 to 28 DPI. Transmission electron microscopy revealed circular virus particles of approximately 25 nm in diameter in the cytoplasm of the villous epithelial cells of the lower small intestine. Histopathological examination did not reveal any abnormalities, such as atrophy, in the intestinal villi. Our results suggest that MuAstV proliferates in the villous epithelial cells of the lower small intestine and has weak pathogenicity.

Creation of an experimental rearing environment for microbiome animal research using an individually ventilated cage system and bioBUBBLE enclosure.

To avoid microbial contamination risk, vinyl film isolators are generally used in animal microbiome experiments involving germ-free (GF) mice and/or gnotobiotic (GB) mice. However, it can take several months to gain expertise in operating the isolator competently. Furthermore, sterilization and sterility testing, which are essential for isolator preparation, can take more than 20 days. Hence, we built an experimental rearing environment that combines an individual ventilation cage system and a bioBUBBLE clean room enclosure to easily set up an experimental animal microbiome environment for animal facilities. In this work, a three-step evaluation was conducted. First, we examined whether GF mice can be maintained in this rearing environment without bacterial contamination. Next, we examined whether GF and GB mice can be maintained without cross-contamination in one individual ventilation cage rack. Finally, we tested whether GF mice can be maintained in a biological safety cabinet controlled by negative pressure. In our series of experiments, no microbial contamination occurred over more than 3 months. These results indicated that our rearing system that combines the individual ventilation cage and bioBUBBLE systems can be used not only for experiments with GF mice but also for Biosafety Level 2 experiments that handle bacteria. Our system can mitigate various disadvantages of using vinyl film isolators. In conclusion, we established an experimental method with improved working time and efficiency compared with those of the previous vinyl isolator method.

Improved engraftment of human peripheral blood mononuclear cells in NOG MHC double knockout mice generated using CRISPR/Cas9.

Humanized mice are widely used to study the human immune system in vivo and develop therapies for various human diseases. Human peripheral blood mononuclear cells (PBMC)-engrafted NOD/Shi-scid IL2rγnull (NOG) mice are useful models for characterization of human T cells. However, the development of graft-versus-host disease (GVHD) limits the use of NOG PBMC models. We previously established a NOG-major histocompatibility complex class I/II double knockout (dKO) mouse model. Although humanized dKO mice do not develop severe GVHD, they have impaired reproductive performance and reduced chimerism of human cells. In this study, we established a novel beta-2 microglobulin (B2m) KO mouse model using CRISPR/Cas9. By crossing B2m KO mice with I-Ab KO mice, we established a modified dKO (dKO-em) mouse model. Reproductivity was slightly improved in dKO-em mice, compared with conventional dKO (dKO-tm) mice. dKO-em mice showed no signs of GVHD after the transfer of human PBMCs; they also exhibited high engraftment efficiency. Engrafted human PBMCs survived significantly longer in the peripheral blood and spleens of dKO-em mice, compared with dKO-tm mice. In conclusion, dKO-em mice might constitute a promising PBMC-based humanized mouse model for the development and preclinical testing of novel therapeutics for human diseases.

Exacerbation of pathogenic Th17-cell-mediated cutaneous graft-versus-host-disease in human IL-1ß and IL-23 transgenic humanized mice.

Although Th17 cells are closely linked to cutaneous graft-versus-host-disease (GVHD) in mouse models, this association remains unclear in human GVHD. In this study, we established a novel xenogeneic cutaneous GVHD model using humanized mice. To induce the differentiation of human Th17 cells, we created transgenic NOG mice expressing human IL-1ß and IL-23 cytokines (hIL-1ß/23 Tg) and transplanted with human CD4+ T cells. The pathologies of cutaneous GVHD, such as a decrease in body weight, alopecia, and T cell inflammation in the skin, were observed much earlier in hIL-1ß/23 Tg mice compared with non-Tg mice after human CD4+ T cell transplantation. In the skin of Tg mice, IL-17- and IFNγ-producing pathogenic Th17 cells were significantly accumulated. Furthermore, high infiltration of murine neutrophils was seen in the skin of Tg mice, but not non-Tg mice, which may have been the cause of the severe alopecia. CD4+ T-cell-transferred hIL-1ß/23 Tg mice were therefore highly sensitive models for inducing cutaneous GVHD mediated by human pathogenic Th17 cells.

A humanized mouse model to study asthmatic airway inflammation via the human IL-33/IL-13 axis.

Asthma is one of the most common immunological diseases and is characterized by airway hyperresponsiveness (AHR), mucus overproduction, and airway eosinophilia. Although mouse models have provided insight into the mechanisms by which type-2 cytokines induce asthmatic airway inflammation, differences between the rodent and human immune systems hamper efforts to improve understanding of human allergic diseases. In this study, we aim to establish a preclinical animal model of asthmatic airway inflammation using humanized IL-3/GM-CSF or IL-3/GM-CSF/IL-5 Tg NOD/Shi-scid-IL2rγnull (NOG) mice and investigate the roles of human type-2 immune responses in the asthmatic mice. Several important characteristics of asthma - such as AHR, goblet cell hyperplasia, T cell infiltration, IL-13 production, and periostin secretion - were induced in IL-3/GM-CSF Tg mice by intratracheally administered human IL-33. In addition to these characteristics, human eosinophilic inflammation was observed in IL-3/GM-CSF/IL-5 Tg mice. The asthmatic mechanisms of the humanized mice were driven by activation of human Th2 and mast cells by IL-33 stimulation. Furthermore, treatment of the humanized mice with an anti-human IL-13 antibody significantly suppressed these characteristics. Therefore, the humanized mice may enhance our understanding of the pathophysiology of allergic disorders and facilitate the preclinical development of new therapeutics for IL-33-mediated type-2 inflammation in asthma.

Long-term maintenance of peripheral blood derived human NK cells in a novel human IL-15- transgenic NOG mouse.

We generated a novel mouse strain expressing transgenic human interleukin-15 (IL-15) using the severe immunodeficient NOD/Shi-scid-IL-2Rγ null (NOG) mouse genetic background (NOG-IL-15 Tg). Human natural killer (NK) cells, purified from the peripheral blood (hu-PB-NK) of normal healthy donors, proliferated when transferred into NOG-IL-15 Tg mice. In addition, the cell number increased, and the hu-PB-NK cells persisted for 3 months without signs of xenogeneic graft versus host diseases (xGVHD). These in vivo-expanded hu-PB-NK cells maintained the original expression patterns of various surface antigens, including NK receptors and killer cell immunoglobulin-like receptor (KIR) molecules. They also contained significant amounts of granzyme A and perforin. Inoculation of K562 leukemia cells into hu-PB-NK-transplanted NOG-IL-15 Tg mice resulted in significant suppression of tumor growth compared with non-transplanted mice. Furthermore, NOG-IL-15 Tg mice allowed for engraftment of in vitro-expanded NK cells prepared for clinical cell therapy. These cells exerted antibody-dependent cell-mediated cytotoxicity (ADCC) on Her2-positive gastric cancer cells in the presence of therapeutic anti-Her2 antibody, and subsequently suppressed tumor growth. Our results collectively suggest that the NOG-IL-15 Tg mice are a useful model for studying human NK biology and evaluating human NK cell-mediated in vivo cytotoxicity.

Incidence of spontaneous lymphomas in non-experimental NOD/Shi-scid, IL-2Rγnull (NOG) mice.

Severely immunodeficient NOD/Shi-scid, IL-2Rγnull (NOG) mice provide an in vivo model for human cell/tissue transplantation studies. NOG mice were established by combining interleukin-2 receptor-γ chain knockout mice and NOD/Shi-scid mice. They exhibit a high incidence of thymic lymphomas and immunoglobulin (Ig) leakiness. In this study, we assessed the incidence of malignant lymphomas and the occurrence of leakiness in 2,184 non-experimental NOG retired breeder mice aged 16-40 weeks. We established that the total incidence of lymphomas was only 0.60% (13/2,184). Most lymphomas (10/13) occurred in female mice by the age of around 25 weeks. No mice developed Ig leakiness. All lymphomas were derived from the thymus, and consisted mainly of CD3-positive and CD45R-negative lymphoblastic-like cells. Therefore, based on the absence of Ig leakiness and a very low incidence of lymphomas, including thymic lymphomas, NOG mice may be useful in regeneration medicine for xenotransplantation of human embryonic stem (ES) cells or induced pluripotent stem (iPS) cells, and in transplantation experiments involving tumor cells.

Study on AAV-mediated gene therapy for diabetes in humanized liver mouse to predict efficacy in humans.

Most in vivo studies on the conversion to insulin-producing cells with AAV carrying PDX1 gene are performed in rodents. However, there is little information regarding Adeno-associated virus (AAV) carrying PDX1 gene transduced to human liver in vivo because accidental death caused by unpredicted factors cannot be denied, such as the hypoglycemic agent troglitazone with hepatic failure. Here we aim to confirm insulin secretion from human liver transduced with AAV carrying PDX1 gene in vivo and any secondary effect using a humanized liver mouse. As the results, AAV2-PG succeeded to improve the hyperglycemia of STZ-induced diabetic humanized liver mice. Then, the analysis of humanized liver mice revealed that the AAV2-PG was more transducible to humanized liver area than to mouse liver area. In conclusion, the humanized liver mouse model could be used to examine AAV transduction of human hepatocytes in vivo and better predict clinical transduction efficiency than nonhumanized mice.

Predominant development of mature and functional human NK cells in a novel human IL-2-producing transgenic NOG mouse.

We generated a severe immunodeficient NOD/Shi-scid-IL-2Rγ(null) (NOG) mouse substrain expressing the transgenic human IL-2 gene (NOG-IL-2 Tg). Upon transfer of human cord blood-derived hematopoietic stem cells (HSCs), CD3(-)CD56(high)CD16(+/-) cells developed unexpectedly, predominantly in the NOG-IL-2 Tg (hu-HSC NOG-IL-2 Tg). These cells expressed various NK receptors, including NKp30, NKp44, NKp46, NKG2D, and CD94, as well as a diverse set of killer cell Ig-like receptor molecules at levels comparable to normal human NK cells from the peripheral blood, which is evidence of their maturity. They produced levels of granzyme A as high as in human peripheral blood-derived NK cells, and a considerable amount of perforin protein was detected in the plasma. Human NK cells in hu-HSC NOG-IL-2 Tg produced IFN-γ upon stimulation, and IL-2, IL-15, or IL-12 treatment augmented the in vitro cytotoxicity. Inoculation of K562 leukemia cells into hu-HSC NOG-IL-2 Tg caused complete rejection of the tumor cells, whereas inoculation into hu-HSC NOG fully reconstituted with human B, T, and some NK cells did not. Moreover, when a CCR4(+) Hodgkin's lymphoma cell line was inoculated s.c. into hu-HSC NOG-IL-2 Tg, the tumor growth was significantly suppressed by treatment with a therapeutic humanized anti-CCR4 Ab (mogamulizumab), suggesting that the human NK cells in the mice exerted active Ab-dependent cellular cytotoxicity in vivo. Taken together, these data suggest that the new NOG-IL-2 Tg strain is a unique model that can be used to investigate the biological and pathological functions of human NK cells in vivo.

NOD-Rag2null IL-2Rγnull mice: an alternative to NOG mice for generation of humanized mice.

We have developed NOD-Rag2(null) IL-2Rγ(null) (NR2G) mice similar to NOD-scidIL-2Rγ(null) (NOG) mice that are known as an excellent host to generate humanized mice. To evaluate the usefulness of NR2G mice as a host for humanized mice, the engraftment rates and differentiation of human cells after human hematopoietic stem cell (HSC) transplantation were compared among NR2G, NOG, and NOD-scid mice. For this purpose, the appropriate irradiation doses to expand the niche for human stem cells in the bone marrow were first determined. As a result, 8 and 2.5 Gy in adult, and 4 and 1 Gy in newborn NR2G and NOG mice, respectively, were found to be appropriate. Next, 5 × 10(4) human umbilical cord blood CD34(+) cells were intravenously inoculated into irradiated adult or newborn of the immunodeficient mice. These HSC transplantation experiments demonstrated that both NR2G and NOG mice showed high engraftment rates compared with NOD-scid mice, although NOG mice showed a slightly higher engraftment rate than that for NR2G mice. However, no difference was found in the human cell populations differentiated from HSCs between NR2G and NOG mice. The HSC transplantation experiments to adults and newborns of two immunodeficient mice also revealed that the HSC transplantation into newborn mice resulted in higher engraftment rate than those into adults. These results showed that NR2G mice could be used as an alternative host to NOG mice to generate humanized mice.

A versatile technique for the in vivo imaging of human tumor xenografts using near-infrared fluorochrome-conjugated macromolecule probes.

Here, we present a versatile method for detecting human tumor xenografts in vivo, based on the enhanced permeability and retention (EPR) effect, using near-infrared (NIR) fluorochrome-conjugated macromolecule probes. Bovine serum albumin (BSA) and two immunoglobulins-an anti-human leukocyte antigen (HLA) monoclonal antibody and isotype control IgG2a-were labeled with XenoLight CF770 fluorochrome and used as NIR-conjugated macromolecule probes to study whole-body imaging in a variety of xenotransplantation mouse models. NIR fluorescent signals were observed in subcutaneously transplanted BxPC-3 (human pancreatic cancer) cells and HCT 116 (colorectal cancer) cells within 24 h of NIR-macromolecule probe injection, but the signal from the fluorochrome itself or from the NIR-conjugated small molecule (glycine) injection was not observed. The accuracy of tumor targeting was confirmed by the localization of the NIR-conjugated immunoglobulin within the T-HCT 116 xenograft (in which the orange-red fluorescent protein tdTomato was stably expressed by HCT 116 cells) in the subcutaneous transplantation model. However, there was no significant difference in the NIR signal intensity of the region of interest between the anti-HLA antibody group and the isotype control group in the subcutaneous transplantation model. Therefore, the antibody accumulation within the tumor in vivo is based on the EPR effect. The liver metastasis generated by an intrasplenic injection of T-HCT 116 cells was clearly visualized by the NIR-conjugated anti-HLA probe but not by the orange-red fluorescent signal derived from the tdTomato reporter. This result demonstrated the superiority of the NIR probes over the tdTomato reporter protein at enhancing tissue penetration. In another xenograft model, patient-derived xenografts (PDX) of LC11-JCK (human non-small cell lung cancer) were successfully visualized using the NIR-conjugated macromolecule probe without any genetic modification. These results suggested that NIR-conjugated macromolecule, preferably, anti-HLA antibody probe is a valuable tool for the detection of human tumors in experimental metastasis models using whole-body imaging.

Establishment of a human allergy model using human IL-3/GM-CSF-transgenic NOG mice.

The development of animal models that mimic human allergic responses is crucial to study the pathophysiology of disease and to generate new therapeutic methodologies. Humanized mice reconstituted with human immune systems are essential to study human immune reactions in vivo and are expected to be useful for studying human allergies. However, application of this technology to the study of human allergies has been limited, largely because of the poor development of human myeloid cells, especially granulocytes and mast cells, which are responsible for mediating allergic diseases, in conventional humanized mice. In this study, we developed a novel transgenic (Tg) strain, NOD/Shi-scid-IL2rγ(null) (NOG), bearing human IL-3 and GM-CSF genes (NOG IL-3/GM-Tg). In this strain, a large number of human myeloid cells of various lineages developed after transplantation of human CD34⁺ hematopoietic stem cells. Notably, mature basophils and mast cells expressing FcεRI were markedly increased. These humanized NOG IL-3/GM-Tg mice developed passive cutaneous anaphylaxis reactions when administered anti-4-hydroxy-3-nitrophenylacetyl IgE Abs and 4-hydroxy-3-nitrophenylacetyl. More importantly, a combination of serum from Japanese cedar pollinosis patients and cedar pollen extract also elicited strong passive cutaneous anaphylaxis responses in mice. Thus, to our knowledge, our NOG IL-3/GM-Tg mice are the first humanized mouse model to enable the study of human allergic responses in vivo and are excellent tools for preclinical studies of allergic diseases.

Expression of apoptosis in placenta of experimental antiphospholipid syndrome mouse.

PROBLEM: To study the histopathology and expression of apoptosis in placenta of pregnancy-complicated antiphospholipid syndrome (APS)-positive mouse models. METHOD OF STUDY: ICR mice were immunized with IgG isotype of human anticardiolipin (aCL) and/or lupus anticoagulant (LA). The pathological and apoptotic expression was studied in the placenta of positive APS mice and compared with respective control samples. RESULTS: Mice with passive immunization of human aCL and/or LA produced an increase in fetal resorption along with markedly induced thrombosis in the placenta associated with increased placental apoptosis. In addition, fewer mitotic cells, less trophoblast giant cell invasion, and more shrunken cells in the deciduas were seen. CONCLUSION: Our study showed the pathogenic role of aCL and LA in pregnancy complications.

Osteosclerosis and inhibition of human hematopoiesis in NOG mice expressing human Delta-like 1 in osteoblasts.

NOD/Shi-scid IL2rγnull (NOG) mice with severe immunodeficiency are excellent recipients to generate "humanized" mice by the transplantation of human CD34(+) hematopoietic stem cells (HSCs). In this study, we developed NOG mice carrying a human Delta-like1 (DLL1) gene, which is a ligand of the Notch receptor and is known to be important in HSC maintenance and self-renewal. We also analyzed the effect of DLL1 signaling on human hematopoiesis and HSC maintenance using humanized DLL1 transgenic NOG mice. To develop DLL1 transgenic NOG (NOG-D1-Tg) mice, a transgenic vector consisting of a human DLL1 complementary DNA fragment placed downstream of the α1(I) collagen (Col1a1) promoter for expression specifically in osteoblasts was constructed. Human CD34(+) HSCs were transplanted into NOG-D1-Tg mice, and differentiation of lymphoid or myeloid lineage cells from human HSCs and maintenance of HSCs in bone marrow were analyzed. Severe osteosclerosis accompanied by increased bone mass and a decreased number of bone marrow cells were observed in NOG-D1-Tg mice. After human HSC transplantation, development of human B lymphocytes, but not T lymphocytes, was significantly suppressed in both bone marrow and the periphery of NOG-D1-Tg mice. Contrary to the initial expectation, retention of human CD34(+) HSCs was inhibited in the bone marrow of NOG-D1-Tg mice. In conclusion, our data suggest that the development of human B lymphocytes and HSC maintenance in osteosclerotic bone may be suppressed by introducing DLL1. These unique humanized mice with sclerotic bone reconstituted by human HSCs are useful models of hematopoiesis in patients with osteosclerosis, such as osteopetrosis, and for investigation of osteogenesis via Notch signaling.

Detection of the onset of ischemia and carcinogenesis by hypoxia-inducible transcription factor-based in vivo bioluminescence imaging.

An animal model for the early detection of common fatal diseases such as ischemic diseases and cancer is desirable for the development of new drugs and treatment strategies. Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that regulates oxygen homeostasis and plays key roles in a number of diseases, including cancer. Here, we established transgenic (Tg) mice that carry HRE/ODD-luciferase (HOL) gene, which generates bioluminescence in an HIF-1-dependent manner and was successfully used in this study to monitor HIF-1 activity in ischemic tissues. To monitor carcinogenesis in vivo, we mated HOL mice with rasH2 Tg mice, which are highly sensitive to carcinogens and are used for short-term carcinogenicity assessments. After rasH2-HOL Tg mice were treated with N-methyl-N-nitrosourea, bioluminescence was detected noninvasively as early as 9 weeks in tissues that contained papillomas and malignant lesions. These results suggest that the Tg mouse lines we established hold significant potential for monitoring the early onset of both ischemia and carcinogenesis and that these lines will be useful for screening chemicals for carcinogenic potential.