IS-Tlk/Kyo, a mutant derived from IS/Kyo strain, exhibits a kinked and/or short tail, in addition to the congenital lumbar vertebral anomaly. Homozygotes of Tlk dominant gene are known to die during embryonic development. We previously reported the morphological features of the skeleton in IS/Kyo and IS-Tlk/Kyo fetuses and of the heart in IS/Kyo fetuses [19]. This study was conducted to clarify the morphological features of the skeleton in both adult rats and of the heart in adult IS/Kyo rats. Ventricular septal defect (VSD) was observed in 3 out of 10 IS/Kyo rats. Neither splitting of lumbar vertebra and supernumerary rib (in both strains) nor fused or absent caudal cartilage (in IS-Tlk/Kyo strain) was detected in adult rats. Fusion of lumbar vertebrae was observed in almost all specimens together with lumbarization of sacral vertebrae in a few specimens in both adult rats as well as fusion of sacral and caudal vertebrae only in adult IS-Tlk/Kyo rats. In addition, a severe reduction in the ossified sacral and caudal vertebrae was noted in adult IS-Tlk/Kyo rats (mean number: 20.6) and IS/Kyo rats (31.8), and the difference was similar to that in the length of sacral and caudal vertebrae. These results suggest that the Tlk gene may be involved in both the congenital and acquired abnormal formation of the lower vertebral centra as well as the persistent occurrence of VSD by the background gene in IS/Kyo strain.

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The aim of this study was to evaluate with anxiety tests the effect of resveratrol (RSV) on streptozotocin (STZ)-induced diabetic mouse behavioral performance at the second and fourth week of treatment. Confirmed diabetic mice (>250 mg/dl of glucose in blood after STZ injection) were treated with RSV (RDM, n=12) or control treated (DM, n=12) for 4 weeks. DM and RDM were tested in the Open Field Test (OFT) and Elevated Plus Maze (EPM). In the second week of RSV treatment, a higher grooming frequency (P<0.05) and a lower defecation and rearing frequency (P<0.05) were detected in the OFT in the RDM group compared with the DM. There was a higher grooming frequency (P<0.05) and higher percentage of entries in open arms (P<0.05) in the RDM group than in the DM group in the EPM. However, in the fourth week of RSV treatment, the only effect observed was a higher grooming frequency in the RDM group than in the DM group (P<0.05) in the EPM. In conclusion, RSV treatment in diabetic mice provoked anxiolytic-like effects in both tests (OFT and EPM), and these effects were observed in a short time window (2 weeks). It is suggested that RSV may help diabetic animals to adapt to new stressing and anxiety situations and thus to improve their welfare.

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Human tumor tissue line models established in the severely immunodeficient NOD.Cg-Prkdc^scid Il2rg^tm1Sug/Jic (NOD/Shi-scid, IL-2Rγ^null or NOG) mouse are important tools for oncology research. During the establishment process, a lymphoproliferative lesion (LPL) that replaces the original tumor cells in the site of transplantation occurs. In the present study, we studied the impact of the LPL on the establishment process and the characteristics of the lesion, investigated the systemic distribution of the lesion in the mouse, and evaluated the potential of a simple identification method. The incidence of the lesion varied among tumor types, and the lesion was found to be the leading cause of unsuccessful establishment with gastric and colorectal cancer. The lesion consisted of a varying population of proliferating lymphoid cells that expressed CD20. The cells were positive for Epstein-Barr virus (EBV)-related antigens, and EBV DNA was detected. There was systemic distribution of the lesion within the NOG mouse, and the most consistent gross finding was splenomegaly. Additionally, identification of LPL-affected cases was possible by detecting splenomegaly in the 1st and 2nd generation mice at necropsy. From our findings the lesion was judged to arise from EBV-infected B cells originating from the donor, and monitoring splenomegaly at necropsy was thought effective as a simple method for identifying the lesion at an early stage of the establishment process.

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To determine the prevalence of drug resistant bacteria colonizing laboratory mice, we isolated and characterized vancomycin-resistant Enterococcus species (VRE) from commercially available mice. A total of 24 VRE isolates were obtained from 19 of 21 mouse strains supplied by 4 commercial breeding companies. Of these, 19 isolates of E. gallinarum and 5 isolates of E. casseliflavus possessing the vanC1 and vanC2/3 genes intrinsically, exhibited intermediate resistance to vancomycin respectively. In addition, these isolates also exhibited diverse resistant patterns to erythromycin, tetracycline, and ciprofloxacin, whereas the use of antibiotics had not been undertaken in mouse strains tested in this study. Although 6 virulence-associated genes (ace, asa, cylA, efaA, esp, and gelE) and secretion of gelatinase and hemolysin were not detected in all isolates, 23 of 24 isolates including the isolates of E. casselifalvus secreted ATP into culture supernatants. Since secretion of ATP by bacteria resident in the intestinal tract modulates the local immune responses, the prevalence of ATP-secreting VRE in mice therefore needs to be considered in animal experiments that alter the gut microflora by use of antibiotics.

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The central region of the primate retina is called macula. The fovea is located at the center of the macula, where the photoreceptors are concentrated to create neural network adapted for high visual acuity. Damage to the fovea by macular dystrophies and age-related macular degeneration (AMD) can reduce the central visual acuity. The molecular mechanisms leading to these diseases are most likely dependent on the proteins in macula differ from that in peripheral retina in expression level. Previously, we reported an early onset macular degeneration with drusen in cynomolgus monkey pedigrees. These monkeys show similar fundus findings of early stage of AMD at 2 years after birth. To elucidate mechanism of drusen formation and to find disease biomarkers for early stage of AMD, we performed plasma proteome analysis. Plasma samples were collected from four affected and control monkeys within the same pedigree. Successful fractionation of the plasma proteins by ProteoMiner and Gelfree8100 were confirmed by SDS-PAGE. Total of 245 proteins were identified from eight samples. From the results of spectral counting, we selected some proteins, Apolipoprotein E, Histidine-rich glycoprotein, and Retinol-binding protein 4 as candidate proteins that would be related with drusen formation. Candidate proteins would be potentially beneficial as biomarkers for human AMD. One of the identified proteins, Apolipoprotein E (ApoE), is structural component of drusen and also related with other neurodegenerative disease like Alzheimer disease. In this plasma proteome analysis, ApoE would be one of the possible factors of early drusen formation in these cynomolgus monkey pedigrees.

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Fluorescent proteins with light wavelengths within the optical window are one of the improvements in in vivo imaging techniques. Near-infrared (NIR) fluorescent protein (iRFP) is a stable, nontoxic protein that emits fluorescence within the NIR optical window without the addition of exogenous substrate. However, studies utilizing an in vivo iRFP model have not yet been published. Here, we report the generation of transgenic iRFP mice with ubiquitous NIR fluorescence expression. iRFP expression was observed in approximately 50% of the offspring from a matings between iRFP transgenic and WT mice. The serum and blood cell indices and body weights of iRFP mice were similar to those of WT mice. Red fluorescence with an excitation wavelength of 690 nm and an emission wavelength of 713 nm was detected in both newborn and adult iRFP mice. We also detected fluorescence emission in whole organs of the iRFP mice, including the brain, heart, liver, kidney, spleen, lung, pancreas, bone, testis, thymus, and adipose tissue. Therefore, iRFP transgenic mice may therefore be a useful tool for various types of in vivo imaging.

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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 × 10⁴ 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.

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Recent studies have suggested the possibility that nocturnal light exposure affects many biological processes in rodents, especially the circadian rhythm, an endogenous oscillation of approximately 24 h. However, there is still insufficient information about the physiological effects of nocturnal light exposure. In this study, we examined the changes in gene expression and serum levels of plasminogen activator inhibitor-1 (PAI-1), a major component of the fibrinolytic system that shows typical circadian rhythmicity, in C3H/He mice. Zeitgeber time (ZT) was assessed with reference to the onset of light period (ZT0). Exposure to fluorescent light (70 lux) for 1 h in the dark period (ZT14) caused a significant increase in hepatic Pai-1 gene expression at ZT16. Serum PAI-1 levels also tended to increase, albeit not significantly. Expression levels of the typical clock genes Bmal1, Clock, and Per1 were significantly increased at ZT21, ZT16, and ZT18, respectively. Exposure to nocturnal light significantly increased plasma adrenalin levels. The effects of nocturnal light exposure on Pai-1 expression disappeared in adrenalectomized mice, although the changes in clock genes were still apparent. In conclusion, our results suggest that nocturnal light exposure, even for 1 h, alters hepatic Pai-1 gene expression by stimulating the adrenal pathway. Adrenalin secreted from the adrenal gland may be an important signaling mediator of the change in Pai-1 expression in response to nocturnal light exposure.

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Genome-wide association studies have revealed that many low-penetrance cancer susceptibility loci are located throughout the genome; however, a very limited number of genes have been identified so far. Using a forward genetics approach to map such loci in a mouse skin cancer model, we previously identified strong genetic loci conferring resistance to chemically induced skin papillomas on chromosome 4 and 7 with a large number of [(FVB/N × MSM/Ms) F₁ × FVB/N] backcross mice. In this report, we describe a combination of congenic mapping and allele-specific alteration analysis of the loci on chromosome 4. We used linkage analysis and a congenic mouse strain, FVB.MSM-Stmm3 to refine the location of Stmm3 (Skin tumor modifier of MSM 3) locus within a physical interval of about 34 Mb on distal chromosome 4. In addition, we used patterns of allele-specific imbalances in tumors from N₂ and N₁₀ congenic mice to narrow down further the region of Stmm3 locus to a physical distance of about 25 Mb. Furthermore, immunohistochemical analysis showed papillomas from congenic mice had less proliferative activity. These results suggest that Stmm3 responsible genes may have an influence on papilloma formation in the two-stage skin carcinogenesis by regulating papilloma growth rather than development.

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Reproductive engineering techniques, such as in vitro fertilization (IVF) and cryopreservation of embryos or spermatozoa, are essential for preservation, reproduction, and transportation of genetically engineered mice. However, it has not yet been elucidated whether these techniques can be applied for the generation of genome-edited mice using engineered nucleases such as transcription activator-like effector nucleases (TALENs). Here, we demonstrate the usefulness of frozen oocytes fertilized in vitro using frozen sperm for TALEN-mediated genome editing in mice. We examined side-by-side comparisons concerning sperm (fresh vs. frozen), fertilization method (mating vs. IVF), and fertilized oocytes (fresh vs. frozen) for the source of oocytes used for TALEN injection; we found that fertilized oocytes created under all tested conditions were applicable for TALEN-mediated mutagenesis. In addition, we investigated whether the ages in weeks of parental female mice can affect the efficiency of gene modification, by comparing 5-week-old and 8–12-week-old mice as the source of oocytes used for TALEN injection. The genome editing efficiency of an endogenous gene was consistently 95–100% when either 5-week-old or 8–12-week-old mice were used with or without freezing the oocytes. Thus, our report describes the availability of freeze-thawed oocytes and oocytes from female mice at various weeks of age for TALEN-mediated genome editing, thus boosting the convenience of such innovative gene targeting strategies.

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