rasH2 mouse: reproducibility and stability of carcinogenicity due to a standardized production and monitoring system.

The rasH2 mouse was developed as a model for carcinogenicity studies in regulatory science. Its phenotype is stable during high-volume production and over successive generations. To produce rasH2 mice, three strains of mice (C57BL/6J-TgrasH2, C57BL/6J, and BALB/cByJ) were maintained individually. Since the homozygous c-HRAS genotype is lethal, hemizygous transgenic mice were maintained by crossing with inbred C57BL/6J mice. After breeding, male B6-transgenic mice were mated with female BALB/cByJ mice to obtain transgenic mice. Pups that were rasH2-Tg (tg/wt) or rasH2-Wt (wt/wt) were confirmed by genotyping. Frozen embryos were preserved by the Central Institute for Experimental Animals (CIEA) and sent to two facilities, CLEA Japan and Taconic Biosciences, where the mice were produced. Production colonies are created in both facilities and supplied to customers worldwide. To prevent genetic drift, the colonies were renewed for up to 10 generations, and renewals were carried out four times every five years from 2005 to 2021. To ensure the uniformity and maintenance of the phenotype of rasH2 mice, the carcinogen susceptibilities were monitored in every renewal of colonies by CIEA based on a standard protocol of the short-term carcinogenicity study using the positive control compound N-methyl-N-nitrosourea (MNU). Furthermore, simple carcinogenicity monitoring targeting the forestomach, the organ most sensitive to MNU, was performed approximately once a year. Based on the optimally designed production and monitoring systems, the quality of rasH2 mice with reproducibility and stability of carcinogenicity is maintained and supplied globally.

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.

SGX523 causes renal toxicity through aldehyde oxidase-mediated less-soluble metabolite formation in chimeric mice with humanized livers.

SGX523 is a c-Met tyrosine kinase inhibitor that failed in clinical trials because of renal toxicity caused by crystal deposits in renal tubules. SGX523 is metabolized by aldehyde oxidase (AOX) in a species-dependent manner to the considerably less soluble 2-quinolinone-SGX523, which is likely involved in the clinically observed obstructive nephropathy. This study investigated the metabolism and renal toxicity of SGX523 in chimeric mice with humanized livers (humanized-liver mice). The 2-quinolinone-SGX523 formation activity was higher in humanized-liver mouse and human hepatocytes than in mouse hepatocytes. Additionally, this activity in the liver cytosolic fraction from humanized-liver mice was inhibited by the AOX inhibitors raloxifene and hydralazine. After oral SGX523 administration, higher maximum concentrations, larger areas under the plasma concentration versus time curves, and higher urinary concentrations of 2-quinolinone-SGX523 were observed in humanized-liver mice than in non-humanized mice. Serum creatinine and blood urea nitrogen levels were elevated in humanized-liver mice following repeated oral SGX523 administration. The accumulation of amorphous material in the tubules and infiltration of inflammatory cells around tubules were observed in the kidneys of humanized-liver mice after repeated oral SGX523 administration. These findings demonstrate that humanized-liver mice are useful for understanding the metabolism and toxicity of SGX523.

[Efficacy of local steroid injection on rat skin lesions induced by extravasation of vesicant anticancer drugs].

The efficacy of local steroid injection on the extravasation of vesicant anticancer drugs is controversial. In this study, the efficacy of local steroid injection was evaluated macroscopically and histologically in the extravasation models of doxorubicin (DXR), vinorelbine (VNR), and paclitaxel (PTX)in rats. Macroscopically, gross skin lesions were reduced by local steroid injections in rats treated with DXR and VNR. PTX did not cause gross skin lesions in most rats regardless of local steroid injection. Histologically, however, DXR, VNR, and PTX all induced deep tissue lesions such as edema, inflammation, and necrosis. Therefore, the effect of local steroid injection seemed to be minimal. In particular, DXR induced extensive necrosis in the subcutaneous and muscle tissues. VNR-induced skin lesions were milder than those induced by DXR, but had full thickness. Lesions caused by PTX were the mildest. These findings suggest that although local steroid injections could serve a primary role in diluting anticancer drugs and reducing gross skin lesions by their anti-inflammatory effect, they have less ability for suppressing deep-tissue lesions developing over time.

Interstitial pneumonia in immunocompetent laboratory rats caused by natural infection with Pneumocystis carinii.

Pneumocystis (P.) carinii is known to cause fatal pneumonia in immunocompromised rats. Cases of P. carinii interstitial pneumonia in immunocompetent rats have been shown histologically to present with perivascular lymphoid cuffs, which have previously been attributed to rat respiratory virus. This study aims to determine the prevalence and pathological characteristics of P. carinii in immunocompetent laboratory rats in experimental facilities in Japan. An epidemiological survey for this agent was performed using PCR to assess 1,981 immunocompetent rats from 594 facilities in Japan. We observed that 6 of the 1,981 rats (0.30%) from 4 out of 594 facilities (0.67%) were positive for P. carinii without infection of other known pathogens. Gross pulmonary lesions were found in 4 of the 6 affected rats. The lungs of these rats contained scattered dark red/gray foci. Histopathologically, the lungs exhibited interstitial pneumonia with lymphoid perivascular cuffs: Pneumocystis cysts were observed using Grocott's methenamine silver stain. To our knowledge, this report is the first to reveal the prevalence of natural P. carinii infection in immunocompetent laboratory rats in Japan.

Clinical and histopathological evaluation of Dermatophagoides farinae-induced dermatitis in NC/Nga mice orally administered Bacillus subtilis.

Probiotic strains have been reported to have the ability to control allergic and inflammatory diseases. In this study, we studied the inhibitory effect of Bacillus subtilis (natto) (BS) on atopic dermatitis. The effects of continuous oral administration of BS for 4 weeks on the development of atopic dermatitis induced by Dermatophagoides farinae body antigen (DF) in NC/Nga (NC) mice were evaluated using 4 groups of mice: group (Gp) DF, DF(+) with no administration of bacteria (n=3); Gp DF/BS, DF(+) and BS(+) (n=5); and Gp PBS, DF(-) with no administration of bacteria (n=3). The mice were gavaged with 1.2 × 10(17) CFU/head of BS 6 times a week for 4 weeks, and DF was applied twice a week for 4 weeks. Histopathological examination revealed significant differences in auricular thickness between Gp DF (664.4 µm, SD=78.0) and Gp DF/BS (278.7 µm, SD = 88.8; p<0.01). The dorsal skin of Gp DF/BS (316.7 µm, SD=187.4) was significantly thinner than that of Gp DF (503 µm, SD=116.3). These results suggest that continuous oral administration of fermented food-derived bacteria (BS) can be effective in alleviating the development of skin lesions induced by DF in NC mice.

Characterization of hiPSC-Derived Muscle Progenitors Reveals Distinctive Markers for Myogenic Cell Purification Toward Cell Therapy.

The transplantation of muscle progenitor cells (MuPCs) differentiated from human induced pluripotent stem cells (hiPSCs) is a promising approach for treating skeletal muscle diseases such as Duchenne muscular dystrophy (DMD). However, proper purification of the MuPCs before transplantation is essential for clinical application. Here, by using MYF5 hiPSC reporter lines, we identified two markers for myogenic cell purification: CDH13, which purified most of the myogenic cells, and FGFR4, which purified a subset of MuPCs. Cells purified with each of the markers showed high efficiency for regeneration after transplantation and contributed to the restoration of dystrophin expression in DMD-immunodeficient model mice. Moreover, we found that MYF5 regulates CDH13 expression by binding to the promoter regions. These findings suggest that FGFR4 and CDH13 are strong candidates for the purification of hiPSC-derived MuPCs for therapeutical application.

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.

Morphological and sequence analysis of Mycoplasma sp. isolated from the oral cavity of a house musk shrew (Suncus murinus).

Mycoplasma sp. strain EDS-4 was isolated from the oral cavity of EDS line of a house musk shrew (Suncus murinus) originated from Bangladesh, and was distinguished from all previously described mollicutes. It lacks a cell wall; ferments glucose; does not produce film and spots; and does not hydrolyse arginine and urea. The strain could be distinguished from all previously described mollicutes by 16S rRNA gene sequence comparisons. The results suggest that the isolate is new species of mollicutes originated from the shrew. The strain EDS-4 has been deposited with Japan collection of Microorganisms, Bioresource Center, RIKEN in Japan (JCM15930). The 16S rRNA gene sequence of strain EDS-4 is available through the DDBJ under accession number (AB469852).

Prevalence of murine astrovirus in laboratory animal facilities in Japan.

To investigate the prevalence of murine astrovirus (MuAstV) in mice in laboratory animal facilities in Japan, a polymerase chain reaction (PCR) test targeting the RNA-dependent RNA polymerase (RdRP) gene was performed on the cecum contents of 1,212 mice (1,183 immunocompetent mice and 29 immunodeficient mice) from 226 facilities. The results showed that 118 (52.2%) of the 226 facilities were positive for MuAstV. Out of the 1,212 mice, 424 (35.0%) were positive. No gross lesions were observed in any of the mice examined. A phylogenetic analysis for 15 selected strains revealed that 13 strains formed one cluster, while two were genetically distant from that cluster. These results suggest that multiple strains are prevalent in laboratory mice in Japan.

Novel gastrointestinal disease in common marmosets characterised by duodenal dilation: a clinical and pathological study.

Common marmosets (Callithrix jacchus) are frequently used for biomedical research but gastrointestinal diseases have been major health problems to maintain captive marmosets. We have diagnosed a novel gastrointestinal disease in marmosets, as which we propose to call 'marmoset duodenal dilation syndrome'; this disease is characterised by proximal duodenal obstruction and dilation. This study aimed to reveal the clinical and pathological findings of this syndrome and establish appropriate diagnostic imaging methods. Animals with the syndrome comprised 21.9% of the necropsy cases at the Central Institute for Experimental Animals in Kawasaki, Japan. The syndrome is characterised by clinical signs included vomiting, bloating, and weight loss. Grossly, all diseased animals exhibited significant dilation of the descending part of the duodenum, which contained a mixture of gas and fluid. The duodenal dilations were definitively diagnosed by contrast radiography. Moreover, a combination of plain radiography and ultrasonography was found to be a viable screening method for diagnosing duodenal dilation. The animals with duodenal dilation characteristically showed adhesions between the descending duodenum and ascending colon with chronic peritonitis. The cause of marmoset duodenal dilation syndrome remains unknown, but was likely multifactorial, including peritoneal adhesion, chronic ulcer, and feeding conditions in this study.

Extracellular nanovesicles for packaging of CRISPR-Cas9 protein and sgRNA to induce therapeutic exon skipping.

Prolonged expression of the CRISPR-Cas9 nuclease and gRNA from viral vectors may cause off-target mutagenesis and immunogenicity. Thus, a transient delivery system is needed for therapeutic genome editing applications. Here, we develop an extracellular nanovesicle-based ribonucleoprotein delivery system named NanoMEDIC by utilizing two distinct homing mechanisms. Chemical induced dimerization recruits Cas9 protein into extracellular nanovesicles, and then a viral RNA packaging signal and two self-cleaving riboswitches tether and release sgRNA into nanovesicles. We demonstrate efficient genome editing in various hard-to-transfect cell types, including human induced pluripotent stem (iPS) cells, neurons, and myoblasts. NanoMEDIC also achieves over 90% exon skipping efficiencies in skeletal muscle cells derived from Duchenne muscular dystrophy (DMD) patient iPS cells. Finally, single intramuscular injection of NanoMEDIC induces permanent genomic exon skipping in a luciferase reporter mouse and in mdx mice, indicating its utility for in vivo genome editing therapy of DMD and beyond.

Study of a Bordetella hinzii isolate from a laboratory mouse.

Bordetella hinzii isolated from the trachea and lungs of a laboratory mouse with a respiratory infection was identified based on its phenotypic and genetic traits. The mouse showed sneezing with a chattering sound but without nasal discharge, and histopathologic examination revealed rhinitis, tracheitis, and bronchopneumonia. The isolate was a gram-negative, oxidase- and catalase-positive, short rod-shaped organism that produced alkali from malonate. The results of biochemical identification, an alkali production test from malonate, and partial sequence analysis of the 16S rRNA gene (1523 bp) were consistent with those reported previously for B. hinzii. The isolate induced sneezing in ICR mice and sneezing and slight to severe dyspnea in NOD-SCID mice after experimental infection. Histopathologic examination revealed catarrhal rhinitis and bronchopneumonia in both strains of mice and interstitial pneumonia in NOD-SCID mice. In light of these findings, B. hinzii was deemed to be a novel causative agent of respiratory disease in mice. This report describes the first isolation of B. hinzii from a mouse and confirms the organism's pathogenicity in mice.

Experimental infection studies of Pasteurella pneumotropica and V-factor dependent Pasteurellaceae for F344-rnu rats.

To investigate the pathogenicities of P. pneumotropica (Pp) and V-factor dependent Pasteurellaceae (VFDP) in immunodeficient rats, experimental infections of F344-rnu rats were performed using 3 strains (ATCC 35149, CNP 160 and RPZ) of Pp and 4 strains (V6, V7, V8 and V9) of VFDP. Four animals per experimental group were inoculated twice on day 0 and post-inoculation day (PID) 14 with bacterial suspension intranasally. Two animals from each group were sacrificed on PID 60 and 120, and examined. In the animals inoculated with strains of Pp, sneezing was observed in some animals inoculated with strains ATCC 35149 and CNP 160 until PID 31. No clinical signs were observed in other animals. The strains were mainly isolated from the nasal cavity and trachea on PID 60, and the nasal cavity, trachea and lung on PID 120. Inflammation and necrosis of nasal cavity mucosa were observed in all animals inoculated with strains ATCC 35149 and CNP 160 in a histopathologic examination. No histopathological changes were observed in any other animal. In the animals inoculated with strains of VFDP, neither clinical disorder nor histopathological change was observed. The strains were mainly isolated from the trachea on PID 60, and from the trachea and lungs on PID 120. From these results, the pathogenicity of Pp in immunodeficient rats appears to differ by strain, and VFDP appears to be non-pathogenic in immunodeficient rats.

Association between Helicobacter pylori infection and platelet count in mice.

Strong evidence for an association between idiopathic thrombocytopenic purpura (ITP) and Helicobacter pylori (HP) infection has been reported in humans. Chronic ITP is known to be improved by the eradication of HP. The purpose of this study was to reproduce these events by the experimental infection of several strains of mice with HP. BALB/c, C57BL/6, and DBA/2 mice were untreated or orally inoculated with HP. Two months later, platelet counts were compared in samples from HP-infected and noninfected mice. Platelet counts (mean ± SD, × 104 cells/µl) in blood samples from HP-infected BALB/c, C57BL/6, and DBA/2 mice were 102.28 ± 14.71, 99.65 ± 17.00, and 111.57 ± 16.20, respectively; the respective counts from noninfected mice were 121.80 ± 13.30, 104.35 ± 18.20, and 107.84 ± 14.33. A significant difference in platelet counts between HP-infected and noninfected mice was observed in BALB/c mice (P≤0.01) but was not observed in DBA/2 mice, even though the histocompatibility (H)-2 type of the DBA/2 was the same as that of BALB/c mice. According to ELISA results, the optical density value for the anti-HP antibody in HP-infected BALB/c mice was not correlated with the number of platelets (P>0.50). These results suggest that the decrease in platelet count caused by HP infection is not related to antibody titer and histocompatibility-2 type. Experimental infection of BALB/c mice with HP can reproduce the relationship between HP and ITP and serves as a good model to investigate the mechanistic basis for the effectiveness of HP eradication therapy for ITP treatment.

Effects of growth factors on development of fetal islet B-cells in vitro.

To investigate the role of growth factors (epidermal growth factor [EGF], betacellulin, and activin A) in the development of islet B cells of rat fetal pancreatic explants in vitro, pancreases from rat fetuses at day 18 of gestation were cultured for 96 hr, with or without these growth factors. Culture medium was changed every 24 hr, and the level of insulin released in the culture medium was measured. After 72 hr of culture, pancreases were examined histologically. As a result, EGF promoted cell proliferation, but reduced B cell volume. Whereas, betacellulin and activin A inhibited cell division, but promoted increased B cell volume and insulin secretion, especially activin A, which stimulated insulin release in a time dependent manner. These results suggest that EGF, betacellulin, and activin A promote pancreatic cell proliferation, islet B-cell differentiation, and islet B-cell differentiation and functional maturation, respectively, and that EGF, betacellulin, and activin A, in this order, regulate islet B-cell neogenesis.

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.

Background data on NOD/Shi-scid IL-2Rγnull mice (NOG mice).

To obtain background data of NOD/Shi-scid IL-2Rγnull (NOG) mice, severely immunedeficient mice, a total of 120 animals were examined at 7, 26 and 52 weeks-old (20 mice/sex/group). The survival rate at 52 weeks-old was 95% (19/20) in both sexes. Clinically, circling behavior in one direction along the cage wall was observed in males after 8 weeks and females after 47 weeks-old, and hunchback position was found in males after 32 weeks-old. Hematologically, lymphocyte count markedly decreased at all ages, while white blood cell count increased in several mice at 52 weeks-old. Blood chemistry results revealed high values of aspartate aminotransferase, lactate dehydrogenase and creatine phosphokinase in some females at 26 weeks-old, without any related histological change. Histologically, lymphoid hypoplasia characterized by severe lymphocyte depletion with poorly developed tissue architectures was observed. In addition, spongiotic change in the nerve tissue was observed in both sexes at 7 and 26 weeks-old, and intracytoplasmic materials known as tubular aggregates in the skeletal muscles were found in males terminated at 26 and 52 weeks-old and in females at 52 weeks-old. Malignant lymphoma was found in one female euthanized at 20 weeks-old. Further, small intestinal adenoma, hepatocellular adenoma, leukemia, cerebral lipomatous hamartoma, Harderian gland adenoma and uterine polyp were also observed, and their incidences were low except for that of uterine polyp. This study provided detailed background data on NOG mice up to 52 weeks-old and provided information on appropriate use of NOG mice in the various research fields.

Small eye phenotypes observed in a human tau gene transgenic rat.

We developed a rat line showing small eye from transgenic rats that were obtained by microinjection of a DNA segment containing the human (h)tau cDNA (GenBank: BC000558: 31-677,774-1180) expressed under control of CAG promoter, which is related to Alzheimer disease, into the pronuclei rat embryos. The rat line was established by selective brother-sister mating of rats showing small eyes. Of 11 offspring in the 11th generation, there were eight animals with microphthalmia and the transgene. The remaining three rats without transgene did not show the small eyes phenotype. The globes of affected rats were 1.2 mm in length compared with normal globes (3.5 mm), and all other ocular structures were normal. The expression of hTau protein was evident immunohistochemically in the ciliary body, extraocular muscle, lens epithelium, and pigment epithelium. Cytogenetic analysis suggested that the chromosome location of the transgene was chromosome 1 (1p12). This region may include genes related to lens development, such as Cat5.

Survey and Experimental Infection of Enteropathogenic Escherichia coli in Common Marmosets (Callithrix jacchus).

Common marmosets (Callithrix jacchus) are frequently used for biomedical research but can be afflicted with diarrhea-a serious and potentially lethal health problem. Enteropathogenic Escherichia coli (EPEC) is thought to be the causative pathogen of hemorrhagic typhlocolitis in common marmosets, but the actual incidence of the disease and the relationship between EPEC and hematochezia are unknown. This study investigated the prevalence of EPEC infection in common marmosets and the association between EPEC and hematochezia. A total of 230 stool or rectal swab samples were collected from 230 common marmosets (98 clinically healthy, 85 diarrhea, and 47 bloody stool samples) and tested by culture-based detection and PCR amplification of VT1, VT2, LT, ST, eae, and bfp genes. Healthy animals were divided into three groups (n = 4 each for high and low concentration groups and n = 2 as negative control), and those in the experimental groups were perorally inoculated with a 2-ml of suspension of EPEC R811 strain adjusted to 5 × 108 (high concentration) and 5 × 104 (low concentration) CFU/ ml. Two animals in each group were examined 3 and 14 days post-inoculation (DPI). EPEC was detected in 10 of 98 clinically healthy samples (10.2%), 17 of 85 diarrhea samples (20%), and all 47 bloody stool samples (100%), with a significant difference detected between presence of EPEC and sample status (P < 0.01). Acute hematochezia was observed in all animals of the high-concentration group but not in other groups at 1 or 2 DPI. A histopathological examination revealed the attachment of gram-negative bacilli to epithelial apical membranes and desquamated epithelial cells in the cecum of animals in the high-concentration group at 3 DPI. These findings suggest that EPEC is a causative agent of hemorrhagic typhlocolitis in common marmosets.