Research

■ Development and Phenotypic Characterization of Renal Disease Model Mice

Chronic kidney disease (CKD) affects approximately one in ten people in Japan and can progress to end-stage renal failure requiring dialysis. There were approximately 340,000 dialysis patients in Japan, making CKD a serious public health issue both medically and economically. This underscores the urgent need for early diagnosis, disease progression control, and the development of new treatments. While CKD has traditionally been attributed to lifestyle-related conditions such as hypertension and diabetes, recent genomic analyses have expanded our understanding by revealing a strong contribution of genetic factors. Some patients diagnosed with CKD have been found to carry mutations associated with hereditary kidney diseases. These variants are now drawing attention as potential hidden risk factors for CKD.

Our team focuses on genetic risk factors that are associated with both hereditary kidney diseases and CKD. Studying these rare inherited disorders can also provide insights into the genetic background of CKD in the general population.

We generate mouse models carrying patient-specific variants using CRISPR/Cas9 genome editing technology. We then perform detailed analyses, including kidney histopathology, urine and blood biochemistry, and gene expression profiling, to investigate the mechanisms of disease onset and progression.

For mouse models that closely recapitulate human disease phenotypes, we collaborate with clinical researchers to expand into drug discovery research. Through this, we aim to establish a strong preclinical foundation for the development of future therapeutic strategies.

Fig.1: Schematic overview of the generation of genome-edited mice using the CRISPR/Cas9 system. Depending on the guide RNA sequence, the Cas9 enzyme introduces a double-strand break at the target gene. During the repair process, a donor DNA sequence carrying a disease-associated variant is incorporated, resulting in a knock-in of the variant into the mouse genome.

Fig.2: Histopathological analysis of mouse glomeruli.
HE (hematoxylin and eosin), PAM, Masson’s trichrome, and PAS staining were used to evaluate the structure of the glomerular basement membrane, mesangial cell proliferation, and tissue fibrosis.

■ Regulatory Role of Tardbp 3′UTR in ALS Pathogenesis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that selectively targets motor neurons, which are nerve cells responsible for controlling voluntary muscles (Fig.3). Motor neuron loss leads to progressive muscle weakness, paralysis, and respiratory failure. Most ALS cases (90–95%) are sporadic, making it difficult to establish robust genetic mouse models that recapitulate the disease. However, abnormal cytoplasmic aggregation of TARDBP (TDP-43) protein is observed in over 97% of ALS patients, regardless of genetic background.

We therefore focus on the mechanisms regulating TDP-43 expression levels. We generated a mouse model with a deletion in the 3´ untranslated region (3´UTR) of Tardbp. This region contains multiple polyadenylation signals and elements involved in autoregulatory feedback through TDP-43 binding.

The deletion of the 3´UTR caused homozygous embryonic lethality after gastrulation due to decreased Tardbp mRNA expression. Aged heterozygous mice exhibit elevated levels of TDP-43 protein in the spinal cord, a reduced number of motor neurons, and mild motor dysfunction (Fig.4). These findings demonstrate that regulatory elements within the Tardbp 3´UTR play a pivotal role in normal development and contribute to TDP-43 pathology relevant to ALS.

Fig.3: Generation and characterization of neurodegenerative mouse models.

Fig.4: Diminished motor neurons in the spinal cord of heterozygous mice. KB staining of the spinal cord sections from wild-type (A) and ALS model (B) mice. (C, D) ChAT expression (magenta) and nuclear staining with DAPI (blue) in the spinal cord. (E) Quantification of motor neuron numbers.

■ Influence of Genetic Diversity on Disease Phenotypes

In addition to lifestyle and environmental factors, genetic variants play a significant role in the development of human diseases. Immune cells involved in inflammation and host defense are known to respond differently depending on genetic background. Understanding these differences is crucial for advancing personalized medicine and drug discovery. However, most laboratory mouse studies rely on a single inbred strain, such as C57BL/6 (B6), making it difficult to model genetic diversity seen in human populations.

To overcome this limitation, our team utilizes the JF1 strain, which originates from the Mus musculus molossinus subspecies and is genetically divergent from the standard reference strains maintained at RIKEN BRC (Fig. 5). By crossing B6 and JF1 mice, we generate F1 hybrids in which both parental alleles coexist within the same cells, allowing direct comparison of cis-regulatory effects in a shared cellular context (Fig.6). Through RNA-seq analysis of macrophages derived from these F1 hybrids, we identified allele-specific expression (ASE) differences in genes involved in immune responses and metabolic pathways. Notably, glycolytic enzymes and cytokine genes exhibited pronounced allelic biases, suggesting potential effects on immune activation and macrophage polarization (M1/M2). We also observed that allelic biases often occurred near structural variants (SVs), and our findings suggest that repetitive elements such as SINEs and LINEs may contribute to cis-regulatory divergence between subspecies.

Looking ahead, we plan to expand our analysis to other cell types to better understand how genetic diversity influences disease risk. By leveraging genomic variation across different mouse subspecies, we aim to build a foundation for more precise disease modeling. Using F1 hybrids from genetically distinct subspecies, our team is identifying cis-regulatory elements that drive phenotypic variations.

Fig.5: Distribution of mouse subspecies. Mus musculus domesticus originates from wild mice in Western Europe, while Mus musculus molossinus originates from wild mice in East Asia.

Fig:6: Overview of allele-specific expression analysis using F1 hybrid mice. Under a shared transcriptional environment, expression levels of B6- and JF1-derived alleles are compared using SNPs to identify allele-specific expression differences.