Experimental Animals最新文献

The major histocompatibility complex (MHC) plays a critical role in individual immune responses and susceptibility to various conditions, including autoimmune diseases and drug reactions. In dogs, the canine MHC (dog leukocyte antigen, DLA) polymorphism is key to understanding immune mechanisms, but technical challenges have impeded its comprehensive genetic analysis. This study addressed these issues by using a novel DLA genotyping method combining long-range PCR and PacBio single-molecule real-time sequencing to analyze the full-length DLA class I and II gene sequences in 83 beagle dogs from two different strains (TOYO and Marshall), which are commonly used as laboratory animals. As a result of genotyping using the full-length sequences, 9, 5, 2, 6, and 8 extended alleles were newly discovered for the DLA class I genes in DLA-88, DLA-12, DLA-88L, DLA-64, and DLA-79, respectively. For the DLA class II genes, 11, 18, 12, and 8 extended alleles were newly discovered in DLA-DRA, DLA-DRB1, DLA-DQA1, and DLA-DQB1, respectively. There were 25 haplotypes consisting of extended alleles, in contrast to only 10 haplotypes classified using only peptide binding site sequences. Furthermore, comparisons between the strains revealed differences in haplotype frequencies and genetic differentiation. The full-length analysis also provided preliminary insights into regulatory elements, such as promoter and CpG island polymorphisms in DLA-DQB1. The results of this research have important implications for the understanding of the relationship between DLA polymorphism at full length and individual immune responses in dogs.

The Cre-loxP system has been widely used in neuroscience for spatial and temporal control of gene expression. Although it is considered a powerful genetic tool, increasing evidence suggests that the Cre recombinase itself, without targeting loxP sites, may affect brain function and behavior. In this study, we assessed behavioral phenotypes in several Cre-driver rat lines, each expressing Cre under a different promoter (CAG, Pvalb, TH, Drd2, Tac1, or Thy1) to determine whether Cre expression influences behavioral outcomes. Behavioral testing included open field (for locomotor activity), hot plate (for nociceptive responses), prepulse inhibition (for sensorimotor gating), and contextual and cued fear conditioning (for associative learning). The Drd2-Cre rats exhibited significantly increased locomotor activity and movement speed compared to wild-type controls, suggesting heightened baseline activity. The CAG-Cre rats spent more time in the center area of the open field and showed reduced freezing during fear conditioning, suggesting changes in emotional regulation or learning. In contrast, no significant differences in the nociceptive responses or prepulse inhibition were observed in any of the Cre-driver lines. Following behavioral testing, we conducted structural MRI scans and found no obvious abnormalities in brain morphology across any of the Cre-driver lines, suggesting that the observed behavioral changes may reflect subtle neural alterations. These results suggested that Cre expression can produce promoter-dependent behavioral alterations, particularly in emotion or cognition. Therefore, behavioral validation of Cre-driver lines is recommended in neural circuit studies.

Uncovering protein interaction networks in vivo is essential for understanding physiological and pathological processes. Here, we report the generation of a novel knock-in mouse model expressing miniTurbo, a highly active biotin ligase, fused to the endogenous Usp46 gene. This model enables proximity-dependent biotin labeling (BioID) of USP46-associated proteins in the brain. In adult mice, biotinylation was induced by feeding a 0.1% biotin diet. We further evaluated whether the combination of miniTurbo and dietary biotin supplementation is effective for BioID in the developing brain. Biotinylation was successfully induced in embryonic and neonatal brains via maternal biotin intake, demonstrating the transfer of biotin to the offspring through the placenta during pregnancy and through milk during lactation. This strategy enables proximity labeling under physiological conditions without invasive procedures, such as repetitive subcutaneous injections, during developmental stages. Using mass spectrometry, we identified USP46-proximal proteins, including known cofactors WDR48 and WDR20, in the adult brain. Gene Ontology analysis revealed enrichment in postsynaptic pathways, consistent with known localization of USP46. Among the identified proteins, PLPP3, a phospholipid phosphatase, was significantly downregulated in the hippocampus of Usp46-knockout mice. These findings establish the USP46-miniTurbo knock-in mouse as a powerful tool for in vivo interactome analysis and provide new insights into the molecular functions of USP46 in the brain.

Alternative educational tools, such as training videos and simulators, are recommended in the education of laboratory animal science. However, evidence supporting their educational utility in the training of rodent experimental techniques remains limited. In this study, we assessed the utility of alternative educational tools in the practice of laboratory animal science for novice veterinary students. 149 students participated in a stepwise program beginning with lectures, followed by preparatory learning sessions using training videos and two types of mouse simulators (a silicone-based model and fabric toy mouse), and then hands-on training with live mice. The program covered basic techniques: habituation, restraint, and vaginal smear sampling for estrous cycle determination. A survey-based evaluation was conducted to assess the educational utility of alternative educational tools. The contribution of each preparatory resource (videos, lectures, simulators, printed materials, and notes) to skill acquisition was evaluated, showing that videos, lectures, and printed materials highly contributed. The training videos were rated as more necessary than the simulators for skill acquisition. Psychological evaluation showed that 84% of students experienced anxiety before practice. A positive correlation was found between anxiety levels and frequency of use for all three tools, and students reported that all tools were effective in reducing anxiety during practice. All techniques showed high proficiency rates. Our findings suggest that integrating alternative tools with live-animal training promotes technical skill acquisition, enhances psychological readiness, and supports 3Rs-based laboratory animal practice.

In 2016, an outbreak of Rattus norvegicus polyomavirus 2 (RatPyV2) infection was reported in a colony of X-linked severe combined immunodeficiency (XSCID) rats in the United States. While RatPyV2 infection persists asymptomatically in immunocompetent rats, immunodeficient XSCID rats develop variable respiratory symptoms, emaciation, impaired breeding performance, and systemic deteriorating condition. RatPyV2 is an epitheliotropic virus targeting epithelial cells of the salivary glands, Harderian glands, extraorbital lacrimal glands, respiratory system, and reproductive or accessory reproductive organs. Histopathologically, the formation of large basophilic nuclear inclusion bodies in the infected epithelial cells is a characteristic feature, along with hyperplasia or dysplasia. Glandular atrophy and loss, accompanied by fibrosis and mononuclear cell infiltration, are also observed in the salivary glands, Harderian glands, and extraocular lacrimal glands. In particular, the parotid salivary glands are prone to be severely and extensively affected with relatively severe and diffuse lesions even at one month of age. Severely affected animals also develop interstitial pneumonia. Among target tissues, the parotid salivary glands appear to be higher susceptible to RatPyV2, therefore pathological examination and PCR examination of the salivary glands, including the parotid salivary glands, are essential for the diagnosis of RatPyV2 infection. This review paper provides a comprehensive summary of the features (clinical signs, pathological findings, and transmission), diagnostic methods, and prevalence of RatPyV2 infection, based on our research and reports from research groups in the United States.This is a secondary publication of "Rat polyomavirus 2 infection" [25] published in JALAS Newsletter (in Japanese) 2021, 70 (1): 17-25. It also includes updated content.

Chronic kidney disease (CKD) is a complicated systemic disease displaying various pathophysiological symptoms including mineral bone disorder (CKD-MBD). Ideally, early intervention for CKD-MBD would be desirable, however, there is not enough evidence regarding treatment of CKD-MBD, especially in its early stages, due to its multifactorial pathophysiology and the difficulty in generating adequate animal models. In this study, we evaluated the efficacy of a tissue nonspecific alkaline phosphatase (TNAP) inhibitor, SBI-425 in a CKD-MBD animal model, produced by a combination of nephrectomy and high inorganic phosphate (P_i) diet. This combination induced renal damage, and significantly elevated blood urea nitrogen (BUN). Plasma levels of fibroblast growing factor 23 (FGF-23), parathyroid hormone (PTH) and phosphate were also elevated, leading to ectopic calcification in the kidneys, particularly in the renal tubules. We orally administered SBI-425 twice daily for 12 weeks at doses of 1 and 10 mg/kg, and this treatment significantly inhibited the progression of calcium deposition in the renal tubules. Furthermore, SBI-425 effectively prevented the deterioration of plasma parameters, BUN, FGF-23, PTH, and phosphate. In conclusion, our findings suggest that TNAP inhibition can effectively slow the progression of CKD-MBD by inhibiting the calcification in the renal tubules. These results may have implications for better clinical care of patients with CKD.

Heart failure (HF) is a clinical syndrome related to multiple causes, including oxidative stress. Acyl-CoA thioesterase 1 (ACOT1) is an enzyme in fatty acids metabolism, but it remains unclear in HF. Transverse aortic coarctation induced HF mouse model and hypoxia-stimulated cardiomyocyte (HL-1) model were established. ACOT1 expression was down-regulated in heart tissues of HF mice. Adeno-associated virus serotype 9 (AAV9)-mediated ACOT1 overexpression improved cardiac function and pathological injury of heart tissues in transverse aortic coarctation (TAC)-induced HF mice. ACOT1 overexpression ameliorated oxidative stress in heart tissues of HF mice and hypoxia-stimulated HL-1 cells, as indicated by reduced reactive oxygen species (ROS) and malondialdehyde (MDA) levels and elevated superoxide dismutase (SOD) and glutathione (GSH) levels. We found that ACOT1 overexpression inhibited apoptosis both in vivo and in vitro, with decreased protein levels of cleaved PARP, cleaved CASPASE-3, and cleaved CASPASE-9. Mechanically, ACOT1 activated Kelch-like ECH-associated protein1-NF-E2-related factor2 (KEAP1-NRF2) pathway, leading to the nuclear translocation of NRF2 and increased NRF2-regulated gene Nqo1 expression. Rescue experiment indicated that ML385 (NRF2 inhibitor) abolished the effect of ACOT1 overexpression on oxidative stress. Collectively, these results suggested that ACOT1 overexpression protects heart from injury by inhibiting oxidative stress and apoptosis, possibly through activating KEAP1-NRF2 pathway.

In vivo imaging enables real-time detection of excitation and emission signals and is useful for the noninvasive evaluation of temporal changes in biological tissues. The near-infrared fluorescent protein iRFP can be used for deep-tissue imaging because it emits light at wavelengths that are less attenuated by biological tissues. However, autofluorescence originating from diet, tissues, and the imaging environment can interfere with fluorescence detection; therefore, appropriate animal pretreatment and optimization of imaging conditions are essential. We generated two mouse strains: Alb^eGiR reporter mice, in which enhanced green fluorescent protein (eGFP) and iRFP713 genes were tandemly inserted downstream of the Albumin gene, and hairless mice (Hr^Δ164/Δ164), carrying a mutation in the hairless gene. Their offspring were used in in vivo imaging experiments to investigate: (i) the localization of eGFP and iRFP713 fluorescence, (ii) the influence of hair on fluorescence detection, and (iii) suitable filter combinations for fluorescence detection. In the resulting mice, liver-specific expression of both eGFP and iRFP713 was observed at the same anatomical location. Although autofluorescence was more prominent in hairless mice than in furred mice, signal detection was improved either by using longer-wavelength excitation/emission filters or by applying spectral unmixing to separate the target signal. These findings provide practical guidance for optimizing in vivo fluorescence imaging conditions using standard IVIS platforms.

Emerging evidence indicates that oxidative stress in skeletal muscle is a prerequisite for sarcopenia in diabetic patients. In this study, we show that ubiquitin-specific protease (USP) 2 mitigates the accumulation of reactive oxygen species (ROS) in mature muscle cells. Treatment with ML364, a canonical USP2 inhibitor, robustly increased mitochondrial ROS in mouse C2C12 myotubes and caused an accompanying increase in the glutathione disulfide (GSSG)/glutathione (GSH) ratio. ML364 also caused mitochondrial damage in C2C12 myotubes, resulting in a reduction in intracellular adenosine triphosphate levels. Correspondingly, under diabetic condition, the muscle-specific Usp2-knockout (msUsp2KO) C57BL/6N mice exhibited a significantly higher lipid peroxide level and GSSG/GSH ratio in skeletal muscle than the control mice. The msUsp2KO mice also exhibited augmented insulin resistance and glucose intolerance, but showed no obvious deterioration in muscle weight or histology relative to the control mice. However, damaged mitochondria in the soleus muscle were more frequently observed in msUsp2KO mice than in the control mice. Together, these data suggest that USP2 mitigates ROS accumulation and subsequent mitochondrial damage in muscle cells in mice.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease that causes the selective loss of motor neurons. A histopathological hallmark of ALS is the cytoplasmic aggregation of TDP-43, a ubiquitously expressed RNA-binding protein involved in transcription and splicing regulation. To prevent abnormal accumulation, TDP-43 controls its expression levels through an autoregulatory feedback loop. While most ALS studies have focused on pathogenic variants that impair the protein function of TDP-43, the mechanisms underlying endogenous TDP-43 dysregulation mediated by non-coding elements, including the 3' untranslated region (3'UTR), remain incompletely understood. In this study, we generated a mouse model carrying a targeted deletion of the Tardbp 3'UTR that encompasses the TDP-binding region, polyadenylation signals, and alternative intronic sequences. Our findings demonstrate that the Tardbp 3'UTR is essential for normal mouse development. Loss of this region led to decreased Tardbp mRNA expression and embryonic lethality after gastrulation. Young heterozygous mice were phenotypically normal with no overt disruption in TDP-43 autoregulation. However, aged heterozygous mice displayed mild locomotor dysfunction accompanied by a modest increase in spinal cord TDP-43 protein levels and a reduction in motor neuron numbers. These findings indicate that regulatory elements within the Tardbp 3'UTR play a pivotal role in normal development and contribute to TDP-43 pathology relevant to ALS.