Search Results (117)

The endocannabinoid system (ECS) is known to regulate crucial bodily functions, including healthy muscle activity. However, its precise roles in normal skeletal muscle function and the development of muscle disorders remain unclear. Previously, we developed a tamoxifen-inducible, skeletal muscle-specific CB₁ receptor knockdown (skmCB1-KD) mouse model using the Cre/LoxP system. In this study, we aimed to clarify the mechanisms behind the observed reduction in muscle force generation in these mice. To investigate this, we analyzed calcium dynamics following electrical stimulation-induced muscle fatigue, assessed store-operated calcium entry (SOCE), and performed functional analysis of mitochondrial respiration. Our findings suggest that the reduced muscle performance observed in vivo likely arises from interconnected alterations in ATP production by mitochondria. Moreover, in skmCB1-KD mice, we detected a significant decrease in a component of the respiratory chain (complex IV) and a slowed dissipation of mitochondrial membrane potential upon the addition of an un-coupler (FCCP). Full article

Background: Sepsis is a life-threatening condition that is characterized by systemic inflammation and organ dysfunction, with adrenal dysfunction being a significant complication. This study aimed to investigate the role of necroptosis and hydrogen sulfide (H₂S) in sepsis-induced adrenal dysfunction. Methods: A cecal ligation and puncture (CLP)-induced sepsis mouse model was employed. Adrenocortical-specific mixed lineage kinase domain-like pseudokinase (MLKL) knockout (MLKL-KO) and cystathioneine β-synthase (CBS) knockout (CBS-KO) mice were generated using Cre-loxP technology and adrenocortical-specific Cre tool mice. In vitro experiments utilized TNFα-stimulated Y1 adrenocortical cells. The treatments included the H₂S donor NaHS, TNFα inhibitor R-7050, necroptosis inhibitor NSA and CBS inhibitor AOAA. Pathological assessment involved hematoxylin–eosin (H&E) staining and a Western blot analysis of necroptosis markers (the phosphorylation of MLKL (p-MLKL) and phosphorylation of receptor-interacting protein kinases 1 (p-RIPK1)). Results: Sepsis induced adrenal congestion, elevated TNFα levels, and activated necroptosis (increased p-MLKL/p-RIPK1) in wild-type mice. H₂S treatment attenuated adrenal damage, reduced TNFα, and suppressed necroptosis. MLKL knockout reduced septic adrenal dysfunction, whereas CBS knockout exacerbated septic adrenal dysfunction. In vitro, TNFα induced Y1 cell necroptosis, which was reversed by H₂S or NSA. AOAA exacerbated TNFα-induced necroptosis in Y1 cells. Conclusions: H₂S inhibits TNFα-mediated necroptosis, thereby preserving adrenal integrity in sepsis. Targeting the TNFα–necroptosis axis and enhancing endogenous H₂S production may represent novel therapeutic strategies for sepsis-associated adrenal dysfunction. Full article

Implantation is a complex and tightly regulated process essential for the establishment of pregnancy. It involves dynamic interactions between a receptive uterus and a competent embryo, orchestrated by ovarian hormones such as estrogen and progesterone. These hormones regulate proliferation, differentiation, and gene expression within the three primary uterine tissue types: myometrium, stroma, and epithelium. Advances in genetic manipulation, particularly the Cre/loxP system, have enabled the in vivo investigation of the role of genes in a uterine compartmental and cell type-specific manner, providing valuable insights into uterine biology during pregnancy and disease. The development of endometrial organoids has further revolutionized implantation research. They mimic the native endometrial structure and function, offering a powerful platform for studying hormonal responses, implantation, and maternal-fetal interactions. Combined with omics technologies, these models have uncovered the molecular mechanisms and signaling pathways that regulate implantation. This review provides a comprehensive overview of uterine-specific genetic tools, endometrial organoids, and omics. We explore how these advancements enhance our understanding of implantation biology, uterine receptivity, and decidualization in reproductive research. Full article

Background/Objectives: The inhibitor of growth family member 3 (ING3) acts as an epigenetic reader through physical interactions with histone-modifying enzymes and subsequent chromatin remodelling processes. It is involved in various cellular functions, such as cell cycle control, cell growth, and apoptosis. Although ING3 was assigned tumour suppressor candidate status in some types of cancers, including prostate cancer, some studies suggest it acts to promote growth. To address these contradictory reports regarding its role in the initiation and progression of prostate cancer, we specifically addressed the question of whether ablation of ING3 in the mouse prostate is sufficient to initiate malignant transformation of the prostate and support its (candidate) tumour suppressor status. Methods: To generate the prostate-specific Ing3 knockout mouse, paternal inheritance of the PB-Cre4 transgene was used, while for the generation of a global knockout control, a female mouse harbouring the PB-Cre4 transgene was utilized. To determine the recombination efficiency of the Cre-LoxP system in the prostate at the Ing3 locus, a duplex probe-based digital PCR assay capable of counting undisrupted Ing3 copies was designed. The impact of DNA recombination on the protein level was investigated by immunohistochemical staining of prostate tissue samples. Results: In the prostate-specific knockout, digital PCR analysis revealed mosaic gene deletion. We found recombination efficiencies in the anterior, dorsolateral, and ventral prostate lobes ranging from approximately 15 to 30%. ING3 staining in the prostate was faint with no detectable differences in signal intensity between the knockout specimen and wild-type controls. This low ING3 expression in the prostate is consistent with observations of X-gal staining of an Ing3-LacZ reporter allele. Immunohistochemistry showed increased expression of DNA-damage-associated markers γH2AX and 53BP1. However, no gross anatomical abnormalities or prostate intraepithelial neoplasia (PIN) lesions in the prostate of tissue-specific knockout animals compared to wild-type controls were observed. Conclusions: Altogether, our data provide evidence that disruption of ING3 expression in prostate cells does not lead to malignant transformation and challenges the idea that ING3 acts primarily in a tumour-suppressive manner. Furthermore, this work supports the crucial role of ING3 in maintaining genomic stability, and we confirmed the embryonic lethal phenotype of homozygous Ing3 null mice that is rescued by ectopic expression of ING3. Full article

Lysine-specific histone demethylase 2 (Kdm2a) is essential for histone modifications involved in development and associated diseases. Nevertheless, the specific functions of Kdm2a in renal development and pathology remain largely unexplored. This study aimed to elucidate the roles of Kdm2a in sustaining the biological functions of the kidney by generating mutant mice with Kdm2a deletion using the Aqp2-cre/Loxp system. Our findings showed that Kdm2a is widely expressed across various mouse tissues, with particularly high expression in the kidney’s cortex and medulla, surpassing that in other tissues. Despite no observable effects on morphology or survival following the conditional knockout of Kdm2a, there was a significant reduction in body weight and bilateral kidney weight compared to controls, most pronounced at the 5-week-old stage (p < 0.05). Post Kdm2a deletion, kidney metabolic functions were impaired, evidenced by altered levels of creatinine, urea, total cholesterol, and low-density lipoprotein. Histological examination revealed that Kdm2a-null kidneys exhibited signs of dysfunction, characterized by macrophage infiltration, fibrosis, inflammatory cell infiltration, and mild thrombosis. Further studies revealed that the expression of chemokine- and pro-inflammatory cytokine-related genes Il-6, Il-8, Tnf-a, and Il-1β was significantly increased in the kidneys of Kdm2a cKO mice compared with controls (p < 0.05). Additionally, the expression of reabsorption-related genes (Aqp-3, Aqp-5, and Aqp-8) was markedly downregulated in Kdm2a-deficient kidneys compared with controls (p < 0.05). Collectively, these findings suggest that Kdm2a is crucial for maintaining kidney function and development, partly through the suppression of inflammation and regulation of gene expression. However, the underlying molecular mechanisms of Kdm2a in kidney development warrant further investigation. Full article

Background/Objectives: Incidence of vector-borne diseases, including rickettsioses and anaplasmosis, has been increasing in many parts of the world. The obligate intracellular nature of rickettsial pathogens has hindered the development of robust genetic tools for the study of gene function and the identification of therapeutic targets. Transposon mutagenesis has contributed to recent progress in the identification of virulence factors in this important group of pathogens. Methods: Combining the efficiency of the himar1 transposon method with a recombinase-mediated system, we aimed to develop a genetic tool enabling the exchange of the transposon with a cassette encoding non-native sequences. Results: This approach was used in Rickettsia parkeri to insert a himar1 transposon encoding fluorescent protein and antibiotic resistance genes for visualization and selection, flanked by mismatched loxP sites to enable subsequent recombinase-mediated cassette exchange (RMCE). RMCE mediated by a plasmid-encoded Cre recombinase was then employed to replace the transposon with a different cassette containing alternate fluorescent and selection markers and epitopes of Anaplasma phagocytophilum antigens. The resulting genetically modified R. parkeri was trialed as a live-attenuated vaccine against spotted fever rickettsiosis and anaplasmosis in mice. Conclusions: The use of this system provides a well-established and relatively efficient way of inserting non-native sequences into the rickettsial genome, with applications for the study of gene function and vaccine development. Full article

Background/Objectives: The objective of this study was to assess the role of a secreted serine protease, kallikrein-related peptidase 6 (KLK6), during colorectal tumorigenesis driven by a mutant Adenomatous polyposis coli (APC) tumor suppressor gene. A first analysis of KLK6 expression in the intestinal tract of Apc-mutant multiple intestinal neoplasia (Apc^Min/+) mice revealed up to four-fold induction of Klk6 mRNA levels in adenomas relative to its level in the adjacent mucosa. Methods and Results: The presence of KLK6 protein in the adenomatous areas was confirmed by immunohistochemistry and optical coherence tomography/laser-induced fluorescence (OCT/LIF) imaging. To assess the contribution of the KLK6 expression on the Apc-mutant intestinal and colon tumorigenesis, we engineered a mouse with floxed alleles of the Klk6 gene (Klk6^lox/lox) and crossed it with a mouse expressing the truncated APC protein under control of the intestinal tract-specific human CDX2P9.5-NLS Cre transgene (CPC;Apc^fl/fl;Klk6^+/+). We found that CPC;Apc^fl/fl mice with disrupted Klk6 gene expression (CPC;Apc^fl/fl;Klk6^fl/fl) had a significantly smaller average size of the small intestinal and colon crypts (p < 0.001 and p = 0.04, respectively) and developed a significantly fewer adenomas (p = 0.01). Moreover, a decrease in high-grade adenomas (p = 0.03) and adenomas with a diameter above 2 mm (p < 0.0001) was noted in CPC;Apc^fl/fl;Klk6^fl/fl mice. Further molecular analysis showed that Klk6 gene inactivation in the small intestine and colon tissues of CPC;Apc^fl/fl;Klk6^fl/fl mice resulted in a significant suppression of transforming growth factor β2 (TGF-β2) protein (p ≤ 0.02) and mitogen-activated protein kinase (MAPK) phosphorylation (p ≤ 0.01). Conclusions: These findings demonstrate the oncogenic role of KLK6 in the mutant Apc-mediated intestinal tumorigenesis and suggest the utility of KLK6 for early diagnosis of colorectal tumors. Full article

Neurodegenerative retinal diseases such as glaucoma, diabetic retinopathy, Leber’s hereditary optic neuropathy (LHON), and dominant optic atrophy (DOA) are marked by progressive death of retinal ganglion cells (RGC). This decline is promoted by structural and functional mitochondrial deficits, including electron transport chain (ETC) impairments, increased oxidative stress, and reduced energy (ATP) production. These cellular mechanisms associated with progressive optic nerve atrophy have been similarly observed in familial dysautonomia (FD) patients, who experience gradual loss of visual acuity due to the degeneration of RGCs, which is thought to be caused by a breakdown of mitochondrial structures, and a disruption in ETC function. Retinal metabolism plays a crucial role in meeting the elevated energetic demands of this tissue, and recent characterizations of FD patients’ serum and stool metabolomes have indicated alterations in central metabolic processes and potential systemic deficits of taurine, a small molecule essential for retina and overall eye health. The present study sought to elucidate metabolic alterations that contribute to the progressive degeneration of RGCs observed in FD. Additionally, a critical subpopulation of retinal interneurons, the dopaminergic amacrine cells, mediate the integration and modulation of visual information in a time-dependent manner to RGCs. As these cells have been associated with RGC loss in the neurodegenerative disease Parkinson’s, which shares hallmarks with FD, a targeted analysis of the dopaminergic amacrine cells and their product, dopamine, was also undertaken. One dimensional (1D) proton (¹H) nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry, and retinal histology methods were employed to characterize retinae from the retina-specific Elp1 conditional knockout (CKO) FD mouse model (Pax6-Cre; Elp1^LoxP/LoxP). Metabolite alterations correlated temporally with progressive RGC degeneration and were associated with reduced mitochondrial function, alterations in ATP production through the Cahill and mini-Krebs cycles, and phospholipid metabolism. Dopaminergic amacrine cell populations were reduced at timepoints P30–P90, and dopamine levels were 25–35% lower in CKO retinae compared to control retinae at P60. Overall, this study has expanded upon our current understanding of retina pathology in FD. This knowledge may apply to other retinal diseases that share hallmark features with FD and may help guide new avenues for novel non-invasive therapeutics to mitigate the progressive optic neuropathy in FD. Full article

Hydronephrosis, the dilation of kidneys due to abnormal urine retention, occurs spontaneously in certain inbred mouse strains. In humans, its occurrence is often attributed to acquired urinary tract obstructions in adults, whereas in children, it can be congenital. However, the genetic factors underlying hydronephrosis pathogenesis remain unclear. We investigated the cause of hydronephrosis by analyzing tetraspanin 7 (Tspan7) gene-modified mice, which had shown a high incidence of hydronephrosis-like symptoms. We found that these mice were characterized by low liver weights relative to kidney weights and elevated blood ammonia levels, suggesting liver involvement in hydronephrosis. Gene expression analysis of the liver suggested that dysfunction of ornithine transcarbamylase (OTC), encoded by the X chromosome gene Otc and involved in the urea cycle, may contribute as a congenital factor in hydronephrosis. This OTC dysfunction may be caused by genomic mutations in X chromosome genes contiguous to Otc, such as Tspan7, or via the genomic manipulations used to generate transgenic mice, including the introduction of Cre recombinase DNA cassettes and cleavage of loxP by Cre recombinase. Therefore, caution should be exercised in interpreting the hydronephrosis phenotype observed in transgenic mice as solely a physiological function of the target gene. Full article

Newcastle disease (ND) is a significant infectious disease in poultry, causing substantial economic losses in developing countries. To control ND, chickens must be vaccinated multiple times a year. In order to develop an improved vaccine that provides long-term protection, the F gene from genotype VII NDV was inserted into the herpesvirus of turkey (HVT) vaccine virus using CRISPR/Cas9-mediated NHEJ repair and Cre/LoxP technology. The immunogenicity and protective efficacy of the resulting recombinant vaccines were evaluated through antibody assays and virus challenge experiments. Two recombinant vaccines, rHVT-005/006-F and rHVT-US2-F, were generated, both exhibiting growth rates comparable with those of HVT in vitro and consistently expressing the F protein. One-day-old specific pathogen-free (SPF) chickens immunized with 2000 PFU/bird of either rHVT-005/006-F or rHVT-US2-F developed robust humoral immunity and were completely protected against challenge with the NDV F48E8 strain at 4 weeks post-vaccination (wpv). Furthermore, a single dose of these vaccines provided sustained protection for at least 52 wpv. Our study identifies rHVT-005/006-F and rHVT-US2-F as promising ND vaccine candidates, offering long-term protection with a single administration. Moreover, HVT-005/006 demonstrates promise for accommodating additional foreign genes, facilitating the construction of multiplex vaccines. Full article

Genetic mutations and chronic inflammation of the colon contribute to the development of colorectal cancer (CRC). Using a murine model of inflammation-induced colon tumorigenesis, we determined how genetic mutations alter colon tumor cell differentiation. Inflammation induced by enterotoxigenic Bacteroides fragilis (ETBF) colonization of multiple intestinal neoplasia (Min^ApcΔ716/+) mice triggers loss of heterozygosity of Apc causing colon tumor formation. Here, we report that the addition of BRAF^V600E mutation (BRAF^F-V600ELgr5^{tm1(Cre/ERT2)Cle}Min^ApcΔ716/+, BLM) or knocking out Msh2 (Msh2^LoxP/LoxPVil1-creMin^ApcΔ716/+, MSH2KO) in the Min model altered colon tumor differentiation. Using single-cell RNA sequencing, we uncovered the differences between BLM, Min, and MSH2KO tumors at a single-cell resolution. BLM tumors showed an increase in differentiated tumor epithelial cell lineages and a reduction in the tumor stem cell population. Interestingly, the tumor stem cell population of BLM tumors had revival colon stem cell characteristics with low WNT signaling and an increase in RevCSC marker gene expression. In contrast, MSH2KO tumors were characterized by an increased tumor stem cell population that had higher WNT signaling activity compared to Min tumors. Furthermore, overall BLM tumors had higher expression of transcription factors that drive differentiation, such as Cdx2, than Min tumors. Using RNA velocity, we identified additional potential regulators of BLM tumor differentiation such as NDRG1. The role of CDX2 and NDRG1 as putative regulators for BLM tumor cell differentiation was verified using organoids derived from BLM tumors. Our results demonstrate the critical connections between genetic mutations and cell differentiation in inflammation-induced colon tumorigenesis. Understanding such roles will deepen our understanding of inflammation-associated colon cancer. Full article

Insulin receptor signaling promotes cell differentiation, proliferation, and growth which are essential for oocyte maturation, embryo implantation, endometrial decidualization, and placentation. The dysregulation of insulin signaling in women with metabolic syndromes including diabetes exhibits poor pregnancy outcomes that are poorly understood. We utilized the Cre/LoxP system to target the tissue-specific conditional ablation of insulin receptor (Insr) and insulin-like growth factor-1 receptor (Igf1r) using an anti-Mullerian hormone receptor 2 (Amhr2) Cre-driver which is active in ovarian granulosa and uterine stromal cells. Our long-term goal is to examine insulin-dependent molecular mechanisms that underlie diabetic pregnancy complications, and our conditional knockout models allow for such investigation without confounding effects of ligand identity, source and cross-reactivity, or global metabolic status within dams. Puberty occurred with normal timing in all conditional knockout models. Estrous cycles progressed normally in Insr^d/d females but were briefly stalled in diestrus in Igf1r^d/d and double receptor (DKO) mice. The expression of vital ovulatory genes (Lhcgr, Pgr, Ptgs2) was not significantly different in 12 h post-hCG superovulated ovaries in knockout mice. Antral follicles exhibited an elevated apoptosis of granulosa cells in Igf1r^d/d and DKO mice. However, the distribution of ovarian follicle subtypes and subsequent ovulations was normal in all insulin receptor mutants compared to littermate controls. While ovulation was normal, all knockout lines were subfertile suggesting that the loss of insulin receptor signaling in the uterine stroma elicits implantation and decidualization defects responsible for subfertility in Amhr2-Cre-derived insulin receptor mutants. Full article

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces direct cytopathic effects, complicating the establishment of low-cytotoxicity cell culture models for studying its replication. We initially developed a DNA vector-based replicon system utilizing the CMV promoter to generate a recombinant viral genome bearing reporter genes. However, this system frequently resulted in drug resistance and cytotoxicity, impeding model establishment. Herein, we present a novel cell culture model with SARS-CoV-2 replication induced by Cre/LoxP-mediated DNA recombination. An engineered SARS-CoV-2 transcription unit was subcloned into a bacterial artificial chromosome (BAC) vector. To enhance biosafety, the viral spike protein gene was deleted, and the nucleocapsid gene was replaced with a reporter gene. An exogenous sequence was inserted within NSP1 as a modulatory cassette that is removable after Cre/LoxP-mediated DNA recombination and subsequent RNA splicing. Using the PiggyBac transposon strategy, the transcription unit was integrated into host cell chromatin, yielding a stable cell line capable of inducing recombinant SARS-CoV-2 RNA replication. The model exhibited sensitivity to the potential antivirals forsythoside A and verteporfin. An innovative inducible SARS-CoV-2 replicon cell model was introduced to further explore the replication and pathogenesis of the virus and facilitate screening and assessment of anti-SARS-CoV-2 therapeutics. Full article

The spatiotemporal expression patterns of genes are crucial for maintaining normal physiological functions in animals. Conditional gene knockout using the cyclization recombination enzyme (Cre)/locus of crossover of P1 (Cre/LoxP) strategy has been extensively employed for functional assays at specific tissue or developmental stages. This approach aids in uncovering the associations between phenotypes and gene regulation while minimizing interference among distinct tissues. Various Cre-engineered mouse models have been utilized in the male reproductive system, including Dppa3-MERCre for primordial germ cells, Ddx4-Cre and Stra8-Cre for spermatogonia, Prm1-Cre and Acrv1-iCre for haploid spermatids, Cyp17a1-iCre for the Leydig cell, Sox9-Cre for the Sertoli cell, and Lcn5/8/9-Cre for differentiated segments of the epididymis. Notably, the specificity and functioning stage of Cre recombinases vary, and the efficiency of recombination driven by Cre depends on endogenous promoters with different sequences as well as the constructed Cre vectors, even when controlled by an identical promoter. Cre mouse models generated via traditional recombination or CRISPR/Cas9 also exhibit distinct knockout properties. This review focuses on Cre-engineered mouse models applied to the male reproductive system, including Cre-targeting strategies, mouse model screening, and practical challenges encountered, particularly with novel mouse strains over the past decade. It aims to provide valuable references for studies conducted on the male reproductive system. Full article

The cerebrovascular endothelial cells with distinct characteristics line cerebrovascular blood vessels and are the fundamental structure of the blood–brain barrier, which is important for the development and homeostatic maintenance of the central nervous system. Cre-LoxP system-based spatial gene manipulation in mice is critical for investigating the physiological functions of key factors or signaling pathways in cerebrovascular endothelial cells. However, there is a lack of Cre recombinase mouse lines that specifically target cerebrovascular endothelial cells. Here, using a publicly available single-cell RNAseq database, we screened the solute carrier organic anion transporter family member 1a4 (Slco1a4) as a candidate marker of cerebrovascular endothelial cells. Then, we generated an inducible Cre mouse line in which a CreERT2-T2A-tdTomato cassette was placed after the initiation codon ATG of the Slco1a4 locus. We found that tdTomato, which can indicate the endogenous Slco1a4 expression, was expressed in almost all cerebrovascular endothelial cells but not in any other non-endothelial cell types in the brain, including neurons, astrocytes, oligodendrocytes, pericytes, smooth muscle cells, and microglial cells, as well as in other organs. Consistently, when crossing the ROSA26^LSL-EYFP Cre reporter mouse, EYFP also specifically labeled almost all cerebrovascular endothelial cells upon tamoxifen induction. Overall, we generated a new inducible Cre line that specifically targets cerebrovascular endothelial cells. Full article