CHO-K1 cells (KCLB No. 10061) were cultured in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin at 37 ℃ in 5% CO₂. For Tas2r108 expression, a chimeric construct was produced by inserting somatostatin receptor 3 and KOZAK sequences at the 5-prime end of the Tas2r108 gene to enhance membrane localization. The gene fragment was cloned into the pcDNA3.1 expression vector using EcoRI and XhoI restriction enzymes (Fig. 1). The structural components and specific primer sequences used for this cloning process, including restriction sites for NheI, EcoRI, and XhoI, are summarized in Table 1. Cells were co-transfected with Tas2r108 and G_α16gust44 (provided by Yonsei University) using X-treme GENE HP reagent (Roche, Switzerland) at a 1:2 ratio in Opti-MEM. Expression was confirmed by RT-PCR prior to imaging. Cells seeded on cover glasses were loaded with 2mM fura2-AM and pluronic F-127 (Molecular Probes, Inc., Eugene, USA) in Ca²⁺ measurement buffer (pH 7.4) for 30 minutes at room temperature. After washing, cells were stabilized in the dark for 30 minutes. Fluorescence was measured using a CCD camera-based imaging system (Roper Scientific, USA) and MetaFluor 6.1 software. Intracellular Ca²⁺ changes (F340/F380) were recorded during perfusion of CHX (5, 50, 500 uM) and DEN (50, 150, 500 uM) at 2 ml/min. Cell viability was confirmed by 1 mM ATP treatment at the end of each session. All reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) except specifically marked.

All experimental procedures were approved by the Kangwon National University Animal Care and Use Committee and the Living Modified Organism Commission (GWNU-2020-24-1). C57BL/6 mice were purchased from Orient Bio Inc. (Seongnam, Korea). Mice were maintained in standard plastic cages at 23 ℃ with a 12:12 hour light/dark cycle at the Kangwon National University Animal Care Facility.

Tas2r108^-/- mice were generated using the CRISPR/Cas9 system (Macrogen Inc., Seoul, Korea). Single guide RNAs (sgRNAs) targeting exons 1 and 2 were validated in vitro using Cas9 protein (Fig. 2A). Validated sgRNA-Cas9 complexes were microinjected into fertilized embryos obtained from pregnant mare serum gonadotropin (Daesung Microbiological Labs, Inc., Uiwang, Korea) and human chorionic gonadotropin (Daesung Microbiological Labs, Inc.)-treated female mice. F0 mosaic mice were crossed with Tas2r108^+/+ mice to produce F1 heterozygotes (Fig. 2B). F2 homozygotes were obtained through intercrossing F1 mice (Fig. 2B. Genotypes were confirmed by PCR of tail DNA and sequencing. The specific primers used for genotyping were as follows: forward primer (5`- TGC TCT TGG AGG AAC AGA TTC-3`) and reverse primer (5`- GTG GCC TTC ATT CAT GGA CT-3`). The PCR products were analyzed to distinguish between wild-type (1,448 bp) and knock-out (602 bp) alleles based on their respective molecular weights (Fig. 2C).

Male Tas2r108^+/+ and Tas2r108^-/- mice were individually housed and tested for 48 hours. Mice were provided with two 10 ml pipettes containing water and a bitter solution (CHX or DEN). Pipette positions were swapped every 24 hours to avoid side preference. Fluid intake was measured every 24 hours. The preference score was calculated as: (Bitter solution intake / Total intake) x 100.

To evaluate the long-term effects of Tas2r108 deficiency, mice were monitored until natural death. BW, blood pressure, and blood glucose were measured every two weeks. Blood pressure was recorded from the tail artery using a non-invasive system, CODA-HT2 (Kent Scientific, Torrington, CT, USA), and glucose was measured from peripheral blood using a glucometer, Accu-Chek (Roche Diagnostics, Rotkreuz, Switzerland).

Plasma leptin levels were measured at 8, 12, 16, 20, and 24 weeks of age. Blood was collected from the jugular vein into heparin-treated tubes and centrifuged at 12,000 rpm for 25 minutes. Separated plasma was stored at -80 ℃. Leptin concentration was determined using a mouse/rat leptin ELISA kit (Cat. No. M1305, Morinaga Institute of Biological Science, Inc., Yokohama, Japan) following the manufacturer's protocol, with absorbance measured at 450 nm.

Data were analyzed using the unpaired t test for comparisons between two groups. For concentration-dependent responses, one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test was performed to determine statistical significance. All results are expressed as mean plus or minus SEM, and p < 0.05 was considered statistically significant.

To verify the molecular function of the receptor, we performed in vitro signaling analysis using CHO-K1 cells. RT-PCR confirmed the expression of Tas2r108 (896 bp) and G_α16gust44 (1,125 bp) in the transfected cells (Fig. 3A). Intracellular Ca²⁺ imaging revealed that Tas2r108-transfected cells exhibited robust Ca²⁺ increases in response to 5 uM and 50 uM CHX (Fig. 3B). In contrast, among the tested concentrations of DEN, only 50 uM elicited a significant response (Fig. 3C). When the Ca²⁺ activity for 50 uM CHX and DEN was compared relative to the ATP-induced response, the activity for 50 uM CHX was significantly higher than that for 50 uM DEN (p < 0.001) (Fig. 3D). These results identify CHX as a primary and potent ligand for Tas2r108.

To investigate the physiological role of Tas2r108, we generated Tas2r108^-/- mice using the CRISPR/Cas9 system. Following the microinjection of the Cas9 protein-sgRNA complex into fertilized embryos, 40 F0 generation mice were obtained. Among them, five mice (two males and three females) were identified as Tas2r108 mosaic founders. These founders were crossed with Tas2r108^+/+ mice to produce F1 heterozygotes, and subsequent intercrossing of F1 mice yielded F2 offspring. The F2 generation consisted of 4 Tas2r108^+/+, 12 Tas2r108+/-, and 6 Tas2r108^-/- mice (Table 2). This distribution followed the expected Mendelian inheritance ratio, confirming the successful establishment of the Tas2r108^-/- line.

The effect of Tas2r108 deficiency on bitter taste perception was evaluated using two-bottle preference tests. Notably, a significant reduction in bitter taste sensitivity was observed in Tas2r108^-/- mice at a lower concentration of CHX (5 μM), where they exhibited significantly higher preference scores compared to Tas2r108^+/+ mice (p < 0.001; Fig. 4A). While both groups exhibited a concentration-dependent avoidance of CHX, the Tas2r108^-/- group showed significantly higher preference scores for 50 uM CHX compared to the Tas2r108^+/+ group(Fig. 4A). In contrast, no significant differences were observed between 50 μM CHX and DEN within the Tas2r108^-/- group (Fig. 4B), suggesting that the loss of Tas2r108 specifically impairs the detection threshold for CHX. These findings demonstrate that Tas2r108 is essential for maintaining high sensitivity to specific bitter ligands like CHX.

Long-term physiological monitoring until natural death revealed that Tas2r108 deficiency significantly extends the lifespan of mice. The average lifespan of Tas2r108^+/+ mice was 472 ± 20 days (67.4 weeks), whereas Tas2r108^-/- mice survived for an average of approximately 81.0 weeks(Fig. 5A and B). This represents a statistically significant lifespan extension of about 14 weeks compared to the Tas2r108^+/+ group (Fig. 5B). Regarding BW, Tas2r108^-/- mice exhibited greater weight gain between 16 and 60 weeks of age (Figs. 5A and C). Notably, both groups showed a characteristic rapid weight loss during the terminal phase of life. No adverse effects on embryonic development or reproductive capacity were observed in the Tas2r108^-/- mice (data not shown).

To assess the metabolic changes associated with the observed BW differences, we measured plasma leptin levels at 8, 12, 16, 20, and 24 weeks. Plasma leptin levels in Tas2r108^-/- mice were significantly higher than those in Tas2r108^+/+ mice at the 12, 16, and 20-week marks (Fig. 6). This increase in leptin levels correlated with the period of significant weight gain observed in the Tas2r108^-/- group. In contrast, no significant differences were found in blood pressure, blood glucose, or total food intake between the two groups (data not shown), suggesting that Tas2r108 may specifically modulate systemic metabolic homeostasis and longevity through the leptin signaling pathway.