Identification of Ppy-lineage cells as a novel origin of pancreatic ductal adenocarcinoma.

The Ppy gene encodes pancreatic polypeptide (PP) secreted by PP- or γ-cells, which are a subtype of endocrine cells localised mainly in the islet periphery. For a detailed characterisation of PP cells, we aimed to establish PP cell lines. To this end, we generated a mouse model harbouring the SV40 large T antigen (TAg) in the Rosa26 locus, which is expressed upon Ppy-promoter-mediated Cre-loxP recombination. Whereas Insulin1-CreERT-mediated TAg expression in beta cells resulted in insulinoma, surprisingly, Ppy-Cre-mediated TAg expression resulted in the malignant transformation of Ppy-lineage cells. These mice showed distorted islet structural integrity at 5 days of age compared with normal islets. CK19+ duct-like lesions contiguous with the islets were observed at 2 weeks of age, and mice developed aggressive pancreatic ductal adenocarcinoma (PDAC) at 4 weeks of age, suggesting that PDAC can originate from the islet/endocrine pancreas. This was unexpected as PDAC is believed to originate from the exocrine pancreas. RNA-sequencing analysis of Ppy-lineage islet cells from 7-day-old TAg+ mice showed a downregulation and an upregulation of endocrine and exocrine genes, respectively, in addition to the upregulation of genes and pathways associated with PDAC. These results suggest that the expression of an oncogene in Ppy-lineage cells induces a switch from endocrine cell fate to PDAC. Our findings demonstrate that Ppy-lineage cells may be an origin of PDAC and may provide novel insights into the pathogenesis of pancreatic cancer, as well as possible therapeutic strategies. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Protein acylations induced by a ketogenic diet demonstrate diverse patterns depending on organs and differ between histones and global proteins.

An essential ketone body, ß-hydroxybutyrate (BOHB), plays various roles in physiological regulations via protein acylations such as lysine acetylation and ß-hydroxybutyrylation. Here, to understand how BOHB systemically regulates acylations from an overarching perspective, we administered a ketogenic diet to mice to increase BOHB concentration and examined acylations. We found that global acetylation and ß-hydroxybutyrylation dramatically increase in various organs except for the brains, where the increase was much smaller than in the other organs. Interestingly, we observe no increase in histone acetylation in the organs where significant global protein acetylation occurs despite a substantial rise in histone ß-hydroxybutyrylation. Finally, we compared the transcriptome data of the mice's liver after the ketogenic diet to the public databases, showing that upregulated genes are enriched in those related to histone ß-hydroxybutyrylation in starvation. Our data indicate that a ketogenic diet induces diverse patterns of acylations depending on organs and protein localizations, suggesting that different mechanisms regulate acylations and that the ketogenic diet is associated with starvation in terms of protein modifications.

Does radical cystectomy have a better prognosis than bladder conservative treatment in the real world?

PURPOSE: To compare the prognosis and quality of life between radical cystectomy and bladder conservative treatment for muscle invasive bladder cancer in the real world. MATERIALS AND METHODS: Patients treated for muscle invasive bladder cancer without metastases were retrospectively evaluated for overall survival, progression-free survival, and rehospitalization. RESULTS: Of the 141 patients, 62 underwent bladder conservative treatment and 79 underwent radical cystectomy. Patients who underwent radical cystectomy had significantly better progression-free survival (HR: 1.83, 95% CI: 1.12-3.00; p < 0.01) and overall survival (HR: 1.82, 95% CI: 0.99-3.34; p = 0.03) than those who underwent conservative treatment. However, there was no significant difference in prognosis between patients who refused to undergo radical cystectomy and those who underwent. In addition, rehospitalization rates for complications and additional treatment were significantly higher in patients who received conservative treatment (69.3% vs. 34.2%; p < 0.01), and the length of hospital stay was also prolonged compared to patients who received radical cystectomy (26 vs. 9 days; p = 0.03). CONCLUSIONS: Overall, conservative treatment had a significantly poorer prognosis than radical cystectomy, but there was no significant difference in prognosis when comparing patients who refused radical cystectomy and received conservative treatment with those who received radical cystectomy. However, hospitalization rates and length of stay were significantly worse for patients who chose conservative treatment, which may lead to a decline in quality of life.

Comprehensive analysis of perioperative hypotension in photodynamic diagnosis-assisted transurethral resection of bladder tumor with 5-aminolevulinic acid.

OBJECTIVE: Intraoperative hypotension remains a serious adverse event of photodynamic diagnosis-assisted transurethral resection of bladder tumor with oral administration of 5-aminolevulinic acid. We conducted a re-analysis of perioperative hypotension in photodynamic diagnosis-assisted transurethral resection of the bladder tumor with oral 5-aminolevulinic acid to ascertain its safety. METHODS: A total of 407 cases who underwent transurethral resection of bladder tumors in our institution were reviewed (274 cases for the PDD group with photodynamic diagnosis and 133 for the white light (WL) group without). A classification of hypotension severity was devised to identify risk factors for clinically troublesome hypotension. The distribution of hypotension severity in each of the PDD and WL groups was compared. Additionally, the patient background and perioperative data by hypotension severity were compared only in the PDD group. RESULTS: More patients with moderate and severe hypotension were noted in the PDD group. The renal function was lower with increasing hypotension severity in the PDD group. More patients on general anesthesia were included in the mild and moderate hypotension group, whereas more patients on spinal anesthesia were included in the severe hypotension group. Furthermore, the frequency of side effects other than hypotension tended to increase with hypotension severity. CONCLUSIONS: Renal function impairment and the other adverse effects of 5-aminolevulinic acid may be risk factors for severe hypotension. Mild or moderate hypotension may be caused by general anesthesia and severe hypotension may be caused by spinal anesthesia. To elucidate specific risk factors, further case-control studies are warranted.

Suppression of androgen receptor signaling induces prostate cancer migration via activation of the CCL20-CCR6 axis.

The suppression of androgen receptor (AR) expression exacerbates the migration potential of prostate cancer. This study identified a previously unrecognized regulation of the AR-controlled pathway that promotes migration potential in prostate cancer cells. Prostate cancer cells that pass through a transwell membrane (mig cells) have a higher migration potential with a decreased AR expression than parental cells. In this study, we aimed to elucidate the mechanism of migration enhancement associated with the suppression of AR signaling. Expression of C-C motif ligand 20 (CCL20) is upregulated in mig cells, unlike in the parental cells. Knockdown of AR with small interfering RNA (siAR) in LNCaP and C4-2B cells increased CCL20 secretion and enhanced the migration of cancer cells. Mig cells, CCL20-treated cells, and siAR cells promoted cell migration with an enhancement of AKT phosphorylation and Snail expression, while the addition of a C-C chemokine receptor 6 (CCR6, the specific receptor of CCL20) inhibitor, anti-CCL20 antibody, and AKT inhibitor suppressed the activation of AKT and Snail. With 59 samples of prostate cancer tissue, CCL20 secretion was profuse in metastatic cases despite low AR expression levels. Snail expression was associated with the expression of CCL20 and CCR6. A xenograft study showed that the anti-CCL20 antibody significantly inhibited Snail expression, thereby suggesting a new therapeutic approach for castration-resistant prostate cancer with the inhibition of the axis between CCL20 and CCR6.

Genome-wide screening for regulators of degradation of insulin secretory granules with a fluorescent reporter.

Insulin is essential in controlling blood glucose levels, and its synthesis and secretion have been well investigated. In contrast, how insulin secretory granules (ISGs) are degraded in pancreatic beta cells remains largely unknown. To clarify the mechanism, we constructed a fluorescent reporter detecting ISG degradation, where EGFP and mCherry are tandemly conjugated to a cytoplasmic region of ZnT8, an ISG membrane-localized protein. Depletion of serum and amino acid stimulated lysosomal ISG degradation detected with the reporter. Next, with MIN6 cells expressing Cas9 and the reporter, we investigated the involvement of conventional Atg5/7-dependent autophagy to show that it is dispensable for the ISG degradation process. Finally, we performed genome-wide screening by enriching the cells lacking the ISG degradation and showed that pathways regulating autophagy are not identified. These results suggest that alternative degradation in lysosomes, instead of conventional autophagy, may be involved in ISG degradation.

Genetic ablation of p62/SQSTM1 demonstrates little effect on pancreatic ß-cell function under autophagy deficiency.

Autophagy is known to play an essential role in intracellular quality control through the degradation of damaged organelles and components. We previously demonstrated that ß-cell-specific autophagy deficient mice, which lack Atg7, exhibited impaired glucose tolerance, accompanied by the accumulation of sequestosome 1/p62 (hereafter referred to as p62). Whereas p62 has been reported to play essential roles in regulating cellular homeostasis in the liver and adipose tissue, we previously showed that ß-cell-specific p62 deficiency does not cause any apparent impairment in glucose metabolism. In the present study, we investigated the roles of p62 in ß cells under autophagy-deficient conditions, by simultaneously inactivating both Atg7 and p62 in a ß-cell specific manner. Whereas p62 accumulation was substantially reduced in the islets of Atg7 and p62 double-deficient mice, glucose tolerance and insulin secretion were comparable to Atg7 single-deficient mice. Taken together, these findings suggest that the p62 accumulation appears to have little effect on ß-cell function under conditions of autophagy inhibition.

Usefulness of serum CCL2 as prognostic biomarker in prostate cancer: a long-term follow-up study.

OBJECTIVE: Prostate-specific antigen is considered the most useful biomarker for prostate cancer, but not in all cases. In a previous study, we have shown that a risk classification combining prostate-specific antigen ≥100 ng/mL and chemokine (CC motif) ligand 2 ≥ 320 pg/mL can predict survivals. We investigated the long-term usefulness of serum chemokine (CC motif) ligand 2 as a complementary biomarker to prostate-specific antigen and developed a novel risk classification system. METHODS: Serum samples were collected from 379 patients who underwent prostate biopsy at Kanazawa University Hospital between 2007 and 2013, and 255 patients with histologically diagnosed prostate cancer were included in this study. We retrospectively examined the efficacy of serum chemokine (CC motif) ligand 2 as a prognostic biomarker. RESULTS: Patients with chemokine (CC motif) ligand 2 ≥ 320 pg/mL exhibited a significantly shorter overall survival, prostate cancer-specific survival and castration-resistant prostate cancer-free survival than those with chemokine (CC motif) ligand 2 < 320 pg/mL. Multivariate analysis was performed to determine whether chemokine (CC motif) ligand 2 was a useful prognostic factor. Independent significant predictors of worse overall survival were prostate-specific antigen ≥ 100 ng/mL, Gleason score ≥ 8 and chemokine (CC motif) ligand 2 ≥ 320 pg/dL. Prognostic predictors of prostate cancer-specific survival or cancer-free survival in multivariate analysis were prostate-specific antigen ≥ 100 ng/mL and Gleason score ≥ 8. A novel risk classification system was created to predict overall survival in patients based on the number of risk factors present (chemokine (CC motif) ligand 2 ≥ 320 pg/mL, prostate-specific antigen ≥ 100 ng/mL, Gleason score ≥ 8). Scores 2 or 3, 1 and 0 indicated Poor, Intermediate and Good risk groups, respectively. CONCLUSIONS: This study demonstrated the utility of serum chemokine (CC motif) ligand 2 level as a predictive biomarker of long-term overall survival in prostate cancer. A novel risk classification system that predicts long-term overall survival based on the combined indications of chemokine (CC motif) ligand 2 level, prostate-specific antigen level and Gleason score may be a useful prognostic tool for prostate cancer.

Rubicon in pancreatic beta cells plays a limited role in maintaining glucose homeostasis following increased insulin resistance.

Autophagy has been reported to play a crucial role in the maintenance of intracellular homeostasis, including in pancreatic beta cells. Rubicon, which interacts with the phosphoinositide 3-kinase (PI3K) complex, through autophagy-related 14 (ATG14), is among the few autophagy regulators that have been reported to inhibit autophagic flux to date and the deletion of Rubicon has been shown to increase autophagic flux. Based on previous results showing a causal relationship between autophagic dysfunction and pancreatic beta-cell impairment, we hypothesized that the deletion of Rubicon in pancreatic beta cells would improve cell integrity and confer protective effects. To test this hypothesis, we first confirmed that Rubicon knockdown (KD) promoted autophagic flux in ßTC3 pancreatic beta-cell line. Next, we generated pancreatic beta-cell-specific Rubicon knockout (ßKO) mice, by administering tamoxifen to Rubiconflox/flox:MIP-Cre-ERT mice, which showed normal glucose tolerance and insulin secretion under a normal chow diet, despite successful gene recombination. We also attempted to increase insulin resistance by feeding the mice with a high-fat diet for an additional 2 months to find little differences among the parameters evaluated for glucose metabolism. Finally, severe insulin resistance was induced with insulin receptor antagonist treatment, which resulted in comparable glucose homeostasis measurements between Rubicon ßKO and control mice. In summary, these results suggest that in pancreatic beta cells, Rubicon plays a limited role in the maintenance of systemic glucose homeostasis.

[Effects of Iterative Reconstruction on Image Quality of Pediatric Body Computed Tomography Images].

This study evaluated the effects of three types of hybrid iterative reconstruction (IR) on image quality of pediatric body computed tomography images. The image quality components evaluated were noise power spectrum (NPS), task-based modulation transfer function (TTF), and system performance function (SPF). As the IR strength was increased while reducing the radiation dose, the NPS increased in a low-frequency range and the TTF decreased in low-contrast regions. In the low-contrast regions, the calculated SPF decreased over the entire frequency range. Alternatively, in the high-contrast regions, the SPF decreased in the low-frequency regions and increased in the high-frequency regions. The radiation dose reduction using the hybrid IR resulted in the deterioration of the image quality in the low-contrast regions and changes in the spatial frequency characteristics in the high-contrast regions.

Enhanced Immunoadsorption on Imprinted Polymeric Microstructures with Nanoengineered Surface Topography for Lateral Flow Immunoassay Systems.

Lateral flow immunoassay devices have revolutionized the style of on-site disease detection and point-of-care testing in the past few decades. The surface nanotopography of a solid substrate is a dominant parameter in the efficiency of antibody immobilization, but precise control over surface roughness has not been fully investigated. Here we presented lateral flow immunoassay platforms with nanometer-scale surface roughness, reproducibly engineered using thermal nanoimprinting lithography, and investigated the effects of surface nanotopography on immunoadsorption and immunoassay performance. We fabricated three types of imprinted polycarbonate sheets with microcone array structures having different degrees of surface roughness using three types of molds fabricated by micromachining or laser ablation. The structures fabricated by laser-ablated nickel mold exhibited numerous bumps measuring several tens of nanometers, which enhanced antibody adsorption. We performed sandwich immunoassays of C-reactive protein in serum samples and achieved highly sensitive detection with a detection limit of ∼0.01 µg mL-1 and a broad dynamic range. The present results provide useful information on the remarkable effect of nanoengineered surfaces on biomolecule adsorption, and the platforms presented here will widen the applicability and versatility of lateral flow immunoassay devices.

Establishment of a system for screening autophagic flux regulators using a modified fluorescent reporter and CRISPR/Cas9.

Autophagy is a mechanism of bulk protein degradation that plays an important role in regulating homeostasis in many organisms. Among several methods for evaluating its activity, a fluorescent reporter GFP-LC3-RFP-LC3ΔG, in which GFP-LC3 is cleaved by ATG4 following autophagic induction and degraded in lysosome, has been used for monitoring autophagic flux, which is the amount of lysosomal protein degradation. In this study, we modified this reporter by exchanging GFP for pHluorin, which is more sensitive to low pH, and RFP to mCherry, to construct pHluorin-LC3-mCherry reporter. Following starvation or mTOR inhibition, the increase of autophagic flux was detected by a decrease of the fluorescent ratio of pHluorin to mCherry; our reporter was also more sensitive to autophagy-inducing stimuli than the previous one. To establish monitoring cells for mouse genome-wide screening of regulators of autophagic flux based on CRISPR/Cas9 system, after evaluating knockout efficiency of clones of Cas9-expressing MEFs, we co-expressed our reporter and confirmed that autophagic flux was impaired in gRNA-mediated knockout of canonical autophagy genes. Finally, we performed genome-wide gRNA screening for genes inhibiting starvation-mediated autophagic flux and identified previously reported genes such as Atgs. Thus, we have successfully established a system for screening of genes regulating autophagic flux with our pHluorin-LC3-mCherry reporter in mice.

Thermally imprinted microcone structure-assisted lateral-flow immunoassay platforms for detecting disease marker proteins.

Although various types of on-site immunoassay platforms have been developed, facile and reliable sample-to-answer immunoassay systems are still under development. In this study, we proposed a lateral-flow immunoassay system utilizing a polymer sheet with microcone array structures fabricated by thermal imprinting. By adding a surfactant to the running/washing buffer, we were able to dispense with complicated chemical modification protocols, which are usually necessary to enhance antibody adsorption. We investigated three types of polymeric materials and confirmed that polycarbonate is most suitable as an imprinted polymer substrate. Experiments using microcone arrays with different distances revealed that the increased surface area with nanometer-scale surface roughness was key to achieving stable immobilization of antibodies. To assess the applicability of this assay to clinical diagnosis, C-reactive protein in a pure buffer and in a serum sample was analyzed as a model, with a ∼0.1 µg mL-1 lower limit of detection. The presented approach would open a new path to the development of immunoassay-based on-site point-of-care testing by virtue of its simplicity of operation, high sensitivity, and versatility.

Efficacy of combined prophylactic use of levofloxacin and isepamicin for transrectal prostate needle biopsy: A retrospective single-center study.

PURPOSE: To assess the efficacy of a combined regimen of levofloxacin (LVFX) plus isepamicin (ISP) as prophylaxis for transrectal ultrasound-guided needle biopsy of the prostate (TRUSP-Bx). MATERIALS AND METHODS: Overall, 562 patients undergoing TRUSP-Bx were included in the present study. All patients were administered a single-dose of oral LVFX (500 mg) in the morning and intravenous ISP (400 mg) 60 min before biopsy. All biopsies were performed via TRUSP-Bx with an 18-gauge needle, and 12-core specimens were routinely obtained. Before initiating antibiotic treatment, urine and blood bacterial cultures were tested to determine the causative microorganisms in the patients with acute bacterial prostatitis. RESULTS: Acute bacterial prostatitis developed in three (0.53%) participants. The incidence rates of acute bacterial prostatitis in the low- and high-risk groups were 0.79% and 0.46%, respectively. These patients showed clinical symptoms of acute bacterial prostatitis 12-24 h after their biopsy. Escherichia coli (E. coli) was isolated in the urine or bladder cultures of all of patients. All three isolates were determined to be LVFX-resistant E. coli, although they had good sensitivity to aminoglycosides, cephalosporins, and carbapenems. All patients were administered antibiotic treatment (cephalosporin or carbapenem) immediately and were treated successfully with no evidence of further disease progression. CONCLUSION: Antibiotic prophylaxis with LVFX plus ISP was effective, resulting in a lower incidence of acute bacterial prostatitis after TRUSP-Bx in both low- and high-risk patients.

Significant suppression of Rayleigh scattering loss in silica glass formed by the compression of its melted phase.

We propose the formation of silica glass with improved optical transport properties by compressing its melted phase with a hot isostatic pressure machine at high pressure and temperature. The lowest Rayleigh scattering loss was obtained for the glass held at 200 MPa and 2073 K for 4 h. The observed loss corresponds to 0.07 dB/Km at 1.55 µm, which is about half of the loss in conventional silica glass fiber. The decrease in the loss was well explained in terms of the decrease in the size of the sub-nanometer-sized structural voids observed by positron annihilation lifetime spectroscopy in silica glass. The achievement of high transparency and strong confinement of light represents a promising result for the development of future fiber-core media.

A seed link connector protruding into the bladder formed a bladder stone.

Introduction: Low-dose-rate brachytherapy is performed for localized prostate cancer. We report the first case of a bladder stone encompassing the seed migrated into the bladder in a patient treated with low-dose-rate brachytherapy. Case presentation: A man was diagnosed with prostate cancer and underwent low-dose-rate brachytherapy. After 2 months, dysuria occurred, and ultrasonography revealed a needle-shaped high-intensity protruding from the prostate into the bladder. Cystoscopy examination found a seed link connector. With the possibility of natural dissolution of the seed link, careful observation was chosen. However, 16 months later, hematuria occurred, and an X-ray revealed a bladder stone encompassing the seed. Compared with the X-ray right after seeding, the seed located near the right bladder neck had fallen. The seed was removed by transurethral bladder lithotripsy. Conclusion: Seeds should be carefully located within the prostate, otherwise a bladder stone may be formed encompassing the seed.

Early chemotherapeutic intervention to avoid thyroidectomy in pediatric Langerhans cell histiocytosis with thyroid involvement accompanying tracheal stenosis: a report of two cases.

Thyroid involvement is rare in pediatric Langerhans cell histiocytosis (LCH). It may cause airway narrowing, leading to acute-onset respiratory distress. Severe cases may require emergent surgical interventions such as thyroidectomy, which should be avoided in children due to higher rates of complication, particularly in infancy. There is currently no consensus on the indications for surgical treatment in LCH with thyroid involvement. In this report, we describe the cases of two children who presented with tracheal stenosis caused by thyroid LCH, both of which were successfully treated by early induction of chemotherapy, and one of which was also treated for a shorter duration. Mutation analysis detected in-frame deletions of BRAF exon 12 in both cases. These cases suggest that timely diagnosis and administration of chemotherapy may alleviate severe airway obstruction and reduce the need for thyroidectomy in pediatric patients with thyroid LCH.

Bone Turnover Markers, n-Terminal Propeptide of Type I Procollagen and Tartrate-Resistant Acid Phosphatase Type 5b, for Predicting Castration Resistance in Prostate Cancer.

Bone is a common site of prostate cancer metastasis. Bone turnover markers n-terminal propeptide of type I procollagen (P1NP) and tartrate-resistant acid phosphatase type 5b (TRACP-5b) are highly sensitive to bone remodeling activity. However, their prognostic significance as markers of prostate cancer is unknown. This study retrospectively examined the usefulness of P1NP and TRACP-5b as prognostic biomarkers. Castration-resistant prostate cancer recurrence-free survival (CFS) was estimated using the Kaplan-Meier method. A predictive model for CFS was constructed using multivariate analysis. This study enrolled 255 patients diagnosed with prostate cancer at Kanazawa University Hospital. The median follow-up was 115.1 months. Patients with both high serum P1NP and TRACP-5b levels, defined as having a poor bone turnover category (BTC), had significantly shorter CFS. Multivariate analysis identified Gleason score, metastasis, and BTC poor as predictors for castration resistance in prostate cancer. Using these three factors, a prognostic model was established, categorizing patients into low-risk (no or one factor) and high-risk (two or three factors) groups. In the low-risk group, the median CFS was not reached, contrasting with 19.1 months in the high-risk group (hazard ratio, 32.23, p < 0.001). Combining P1NP and TRACP-5b may better predict castration resistance.

Procalcitonin in advanced urological cancer-bacterial versus non-bacterial infections: prospective cohort study.

OBJECTIVES: Patients with advanced cancer may develop bacterial infections (BI) as their general condition worsens, but general blood tests often find it difficult to distinguish them from non-bacterial infections (NBI). The present prospective study was undertaken to investigate the effectiveness of serum procalcitonin levels in distinguishing between BI and NBI in patients with advanced urological cancer. METHODS: This study prospectively evaluated patients diagnosed with locally advanced or metastatic or recurrent urological cancer in our department from September 2013 to December 2019. Body temperature was measured in the axilla and the measurement results were recorded. Febrile episodes of ≥38.0°C were analysed, and written patient consent was obtained at the onset of the fever. RESULTS: Of 75 patients enrolled in the present study, 90 febrile episodes were analysed. A total of 34 of 90 febrile episodes were regarded as BI, and the remaining 56 febrile episodes as NBI. The median procalcitonin value was significantly higher in the BI group (p=0.0015), while no significant difference was found between the two groups for white blood cell count and C reactive protein. Additionally, a white blood cell count of less than 1.0×10ˆ9/L resulted in BI in all cases. The procalcitonin receiver operating characteristic area under the curve was 0.710 (95% CI 0.586 to 0.83), excluding cases with white blood cell counts of <1.0 × 103/µL. CONCLUSIONS: Procalcitonin is a rapid and affordable marker for differentiation between BI and NBI in patients with advanced urological cancer.

Establishment of Pancreatic ß-Cell-Specific Gene Knockout System Based on CRISPR-Cas9 Technology With AAV8-Mediated gRNA Delivery.

The Cre-loxP system provides valuable resources to analyze the importance of tissue-specific gene knockout (KO), including pancreatic ß-cells associated with the pathogenesis of diabetes. However, it is expensive and time consuming to generate transgenic mice harboring floxed genes of interest and cross them with cell-specific Cre expression mice. We establish a ßCas9 system with mice expressing Cas9 in pancreatic ß-cells and adeno-associated virus 8 (AAV8)-mediated guide RNA (gRNA) delivery based on CRISPR-Cas9 technology to overcome those shortcomings. Interbreeding CAG-loxP-STOP-loxP (LSL)-Cas9 with Ins1-Cre mice generates normal glucose-tolerant ßCas9 mice expressing Cas9 with fluorescent reporter EGFP specifically in ß-cells. We also show significant ß-cell-specific gene KO efficiency with AAV8-mediated delivery of gRNA for EGFP reporter by intraperitoneal injection in the mice. As a proof of concept, we administered AAV8 to ßCas9 mice for expressing gRNA for Pdx1, a culprit gene of maturity-onset diabetes of the young 4. As reported previously, we demonstrate that those mice show glucose intolerance with transdifferentiation of Pdx1 KO ß-cells into glucagon-expressing cells. We successfully generated a convenient ß-cell-specific gene KO system with ßCas9 mice and AAV8-mediated gRNA delivery.