Gene Knockout Mouse Models Research Articles

FBXO22 inhibits colitis and colorectal carcinogenesis by regulating the degradation of the S2448-phosphorylated form of mTOR

Inflammatory bowel disease (IBD) is a considerable threat to human health with a significant risk for colorectal cancer (CRC). However, currently, both the molecular pathogenesis and therapeutic treatment of IBD remain limited. In this report, using both systemic and intestinal epithelium-specific gene knockout mouse models, we demonstrate that FBXO22, a substrate receptor within the SKP1-Cullin 1-F-box family of E3 ubiquitin ligases, plays an inhibitory role in the Azoxymethane/Dextran Sodium Sulfate-induced colorectal inflammatory responses and CRC. FBXO22 targets the serine 2448-phosphorylated form of mammalian mechanistic target of rapamycin (pS2448-mTOR) for ubiquitin-dependent degradation. This proteolytic targeting effect is established based on multiple lines of evidence including the results of colon tissue immunoblots, analysis of cultured cells with altered abundance of FBXO22 by depletion or overexpression, comparison of protein decay rate, effects on mTOR substrates S6K1 and 4E-BP1, analysis of protein-protein interactions, phosphor-peptide binding and competition, as well as reconstituted and cellular ubiquitination. Finally, we have shown that mTOR inhibitor rapamycin (RAPA) was able to alleviate the effects of fbxo22 deletion on colorectal inflammatory response and CRC. These RAPA effects are correlated with the ability of RAPA to inhibit pS2448-mTOR, pS6K1, and p4E-BP1. Collectively, our data support a suppressive role for FBXO22 in colorectal inflammation signaling and CRC initiation by targeting pS2448-mTOR for degradation.

Voltage-gated potassium channels and genetic epilepsy.

Recent advances in exome and targeted sequencing have significantly improved the aetiological diagnosis of epilepsy, revealing an increasing number of epilepsy-related pathogenic genes. As a result, the diagnosis and treatment of epilepsy have become more accessible and more traceable. Voltage-gated potassium channels (Kv) regulate electrical excitability in neuron systems. Mutate Kv channels have been implicated in epilepsy as demonstrated in case reports and researches using gene-knockout mouse models. Both gain and loss-of-function of Kv channels lead to epilepsy with similar phenotypes through different mechanisms, bringing new challenges to the diagnosis and treatment of epilepsy. Research on genetic epilepsy is progressing rapidly, with several drug candidates targeting mutated genes or channels emerging. This article provides a brief overview of the symptoms and pathogenesis of epilepsy associated with voltage-gated potassium ion channels dysfunction and highlights recent progress in treatments. Here, we reviewed case reports of gene mutations related to epilepsy in recent years and summarized the proportion of Kv genes. Our focus is on the progress in precise treatments for specific voltage-gated potassium channel genes linked to epilepsy, including KCNA1, KCNA2, KCNB1, KCNC1, KCND2, KCNQ2, KCNQ3, KCNH1, and KCNH5.

Pleiotropic role of CCR9/CCL25 signaling in adriamycin-induced cardiomyopathy

IntroductionAdriamycin (ADR)-induced cardiomyopathy is a common problem in many cancer survivors. Recently, specific chemokine receptors have garnered interest as therapeutic targets in cardiovascular diseases. ObjectivesThis study aim to report the role of C–C chemokine receptor 9 (CCR9)/C–C chemokine ligand 25 (CCL25) and its therapeutic potential in ADR-induced cardiomyopathy. MethodsFunctional gene knockout and overexpression mouse models were utilized to investigate the role of CCR9 against ADR-induced cardiomyopathy. Transcriptome sequencing was also performed to identify the downstream molecular mechanisms of CCR9. ResultsThis study revealed that CCR9 and CCL25 levels were increased in mice and HL-1 cells injured by ADR, consistent with the results of patients with heart failure. Both in vivo and in vitro, CCR9 overexpression overtly aggravated cardiac dysfunction, accompanied by decreased AMPK activity and increased mitochondrial dysfunction, fibrosis, oxidative stress, and apoptosis. However, the cardiac harmful effects of ADR were reserved by CCR9 knockdown, as well as CCR9 overexpression aggravated cardiotoxicity were reserved by AMPK agonist GSK621. By constructing different domain-missing CCR9 mutants, we suspected that the △4 region of CCR9 is important for AMPK activity. Furthermore, transcriptome sequencing further illustrated the mechanism of CCR9 overexpression aggravated ADR-induced cardiotoxicity, which was associated with CYP1A1. Finally, lithospermic acid (LA) was screened and alleviated ADR-induced cardiotoxicity through regulation of CCR9/CCL25-AMPK signaling, bolstering CCR9-targeted potential clinical application. ConclusionThese findings present a promising target and drug for treating chemotherapy-induced cardiotoxicity.

CRISPR-Mediated Construction of Gene-Knockout Mice for Investigating Antiviral Innate Immunity.

With the rapid development of CRISPR-Cas9 technology, gene editing has become a powerful tool for studying gene function. Specifically, in the study of the mechanisms by which natural immune responses combat viral infections, gene knockout mouse models have provided an indispensable platform. This article describes a detailed protocol for constructing gene knockout mice using the CRISPR-Cas9 system. This field focuses on the design of single-guide RNAs (sgRNAs) targeting the antiviral immune gene cGAS, embryo microinjection, and screening and verification of gene editing outcomes. Furthermore, this study provides methods for using cGAS gene knockout mice to analyze the role of specific genes in natural immune responses. Through this protocol, researchers can efficiently generate specific gene knockout mouse models, which not only helps in understanding the functions of the immune system but also offers a powerful experimental tool for exploring the mechanisms of antiviral innate immunity.

ITGB6 modulates resistance to anti-CD276 therapy in head and neck cancer by promoting PF4+ macrophage infiltration

Enoblituzumab, an immunotherapeutic agent targeting CD276, shows both safety and efficacy in activating T cells and oligodendrocyte-like cells against various cancers. Preclinical studies and mouse models suggest that therapies targeting CD276 may outperform PD1/PD-L1 blockade. However, data from mouse models indicate a significant non-responsive population to anti-CD276 treatment, with the mechanisms of resistance still unclear. In this study, we evaluate the activity of anti-CD276 antibodies in a chemically-induced murine model of head and neck squamous cell carcinoma. Using models of induced and orthotopic carcinogenesis, we identify ITGB6 as a key gene mediating differential responses to anti-CD276 treatment. Through single-cell RNA sequencing and gene-knockout mouse models, we find that ITGB6 regulates the expression of the tumor-associated chemokine CX3CL1, which recruits and activates PF4+ macrophages that express high levels of CX3CR1. Inhibition of the CX3CL1-CX3CR1 axis suppresses the infiltration and secretion of CXCL16 by PF4+ macrophages, thereby reinvigorating cytotoxic CXCR6+ CD8+ T cells and enhancing sensitivity to anti-CD276 treatment. Further investigations demonstrate that inhibiting ITGB6 restores sensitivity to PD1 antibodies in mice resistant to anti-PD1 treatment. In summary, our research reveals a resistance mechanism associated with immune checkpoint inhibitor therapy and identifies potential targets to overcome resistance in cancer treatment.

Selenomethionine supplementation mitigates fluoride-induced liver apoptosis and inflammatory reactions by blocking Parkin-mediated mitophagy in mice

As an environmental pollutant, fluoride-induced liver damage is directly linked to mitochondrial alteration and oxidative stress. Selenium's antioxidant capacity has been shown to alleviate liver damage. Emerging research proves that E3 ubiquitin ligase Park2 (Parkin)-mediated mitophagy may be a therapeutic target for fluorosis. The current study explored the effect of diverse selenium sources on fluoride-caused liver injury and the role of Parkin-mediated mitophagy in this intervention process. Therefore, this study established a fluoride-different selenium sources co-intervention wild-type (WT) mouse model and a fluoride-optimum selenium sources co-intervention Parkin gene knockout (Parkin−/−) mouse model. Our results show that selenomethionine (SeMet) is the optimum selenium supplementation form for mice suffering from fluorosis when compared to sodium selenite and chitosan nano‑selenium because mice from the F-SeMet group showed more closely normal growth and development levels of liver function, antioxidant capacity, and anti-inflammatory ability. Explicitly, SeMet ameliorated liver inflammation and cell apoptosis in fluoride-toxic mice, accomplished through downregulating the mRNA and protein expression levels associated with mitochondrial fusion and fission, mitophagy, apoptosis, inflammatory signalling pathway of nuclear factor-kappa B (NF-κB), reducing the protein expression levels of PARKIN, PTEN-induced putative kinase1 (PINK1), SQSTM1/p62 (P62), microtubule-associated protein light chain 3 (LC3), cysteinyl aspartate specific proteinase 3 (CASPAS3), as well as restraining the content of interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor α (TNF-α), and interferon-γ (IFN-γ). The Parkin−/− showed comparable positive effects to the SeMet in the liver of fluorosis mice. The structure of the mitochondria, mRNA, protein expression levels, and the content of proinflammatory factors in mice from the FParkin−/− and F + SeMetParkin−/− groups closely resembled those in the F + SeMetWT group. Overall, the above results indicated that SeMet could alleviate fluoride-triggered inflammation and apoptosis in mice liver via blocking Parkin-mediated mitophagy.

Eighteen genes primarily expressed in the testis are not required for male fertility in mice†.

There are approximately 20000 protein-coding genes in humans and mice. More than 1000 of these genes are predominantly expressed in the testis or are testis-specific and thought to play an important role in male reproduction. Through the production of gene knockout mouse models and phenotypic evaluations, many genes essential for spermatogenesis, sperm maturation, and fertilization have been discovered, greatly contributing to the elucidation of their molecular mechanisms. On the other hand, there are many cases in which single-gene knockout models do not affect fertility, indicating that tissue-specific genes are not always critical. Here, we selected 18 genes whose mRNA expression is restricted to the testis or higher than in other tissues, but whose function in male reproduction is unknown. We then created single-gene KO mouse models using the CRISPR/Cas9 system. The established KO males were subjected to mating tests and screened for effects on fecundity, revealing that these genes were not essential for spermatogenesis and male fertility. This knowledge will contribute to understanding the functions of genes characteristic of the testis and identify the cause of male infertility.

Anti-Inflammatory Responses Produced with Nippostrongylus brasiliensis-Derived Uridine via the Mitochondrial ATP-Sensitive Potassium Channel and Its Anti-Atherosclerosis Effect in an Apolipoprotein E Gene Knockout Mouse Model.

Atherosclerosis (AS) has become the leading cause of cardiovascular disease worldwide. Our previous study had observed that Nippostrongylus brasiliensis (Nb) infection or its derived products could inhibit AS development by inducing an anti-inflammatory response. We performed a metabolic analysis to screen Nb-derived metabolites with anti-inflammation activity and evaluated the AS-prevention effect. We observed that the metabolite uridine had higher expression levels in mice infected with the Nb and ES (excretory-secretory) products and could be selected as a key metabolite. ES and uridine interventions could reduce the pro-inflammatory responses and increase the anti-inflammatory responses in vitro and in vivo. The apolipoprotein E gene knockout (ApoE-/-) mice were fed with a high-fat diet for the AS modeling. Following the in vivo intervention, ES products or uridine significantly reduced serum and liver lipid levels, alleviated the formation of atherosclerosis, and reduced the pro-inflammatory responses in serum or plaques, while the anti-inflammatory responses showed opposite trends. After blocking with 5-HD (5-hydroxydecanoate sodium) in vitro, the mRNA levels of M2 markers were significantly reduced. When blocked with 5-HD in vivo, the degree of atherosclerosis was worsened, the pro-inflammatory responses were increased compared to the uridine group, while the anti-inflammatory responses decreased accordingly. Uridine, a key metabolite from Nippostrongylus brasiliensis, showed anti-inflammatory and anti-atherosclerotic effects in vitro and in vivo, which depend on the activation of the mitochondrial ATP-sensitive potassium channel.

Mouse models to explore the biological and organismic role of DNA polymerase beta.

Gene knock-out (KO) mouse models for DNA polymerase beta (Polβ) revealed that loss of Polβ leads to neonatal lethality, highlighting the critical organismic role for this DNA polymerase. While biochemical analysis and gene KO cell lines have confirmed its biochemical role in base excision repair and in TET-mediated demethylation, more long-lived mouse models continue to be developed to further define its organismic role. The Polb-KO mouse was the first of the Cre-mediated tissue-specific KO mouse models. This technology was exploited to investigate roles for Polβ in V(D)J recombination (variable-diversity-joining rearrangement), DNA demethylation, gene complementation, SPO11-induced DNA double-strand break repair, germ cell genome stability, as well as neuronal differentiation, susceptibility to genotoxin-induced DNA damage, and cancer onset. The revolution in knock-in (KI) mouse models was made possible by CRISPR/cas9-mediated gene editing directly in C57BL/6 zygotes. This technology has helped identify phenotypes associated with germline or somatic mutants of Polβ. Such KI mouse models have helped uncover the importance of key Polβ active site residues or specific Polβ enzyme activities, such as the PolbY265C mouse that develops lupus symptoms. More recently, we have used this KI technology to mutate the Polb gene with two codon changes, yielding the PolbL301R/V303R mouse. In this KI mouse model, the expressed Polβ protein cannot bind to its obligate heterodimer partner, Xrcc1. Although the expressed mutant Polβ protein is proteolytically unstable and defective in recruitment to sites of DNA damage, the homozygous PolbL301R/V303R mouse is viable and fertile, yet small in stature. We expect that this and additional targeted mouse models under development are poised to reveal new biological and organismic roles for Polβ.

Study based on the acetaldehyde dehydrogenase 2 gene polymorphism and acetaminophen-induced liver injury

Objective: To explore the association between aldehyde dehydrogenase 2 (ALDH2) gene polymorphisms and abnormal liver function-induced by acetaminophen (APAP) drugs. Methods: An ALDH2 gene knockout mouse model was constructed using CRISPR/Cas9 gene editing technology. The obtained heterozygous mice were mated with opposite sex of heterozygotes. Genomic DNA was extracted from the tail of the offspring mouse. The polymerase chain reaction (PCR) method was used to determine the ALDH2 genotype. APAP was further used to induce acute drug-induced liver injury models in wild-type and ALDH2 knockout mice. Blood and liver tissues of mice were collected for liver function index, HE staining, F4/80 immunohistochemistry, and other detections. The intergroup mean was compared using a one-way ANOVA. The LSD- t test was used for pairwise comparison. Results: ALDH2 knockout mice were bred successfully. The genotyping of the offspring was segregated into the wild-type (ALDH2(+/+)), heterozygous mutant (ALDH2(+/-)), and homozygous mutant (ALDH2(-/-)), respectively. Biochemical and histological results after APAP modeling showed that the level of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and total bilirubin (TBil) was not significantly increased in the blank control group (P < 0.05), while the ALT, AST,ALP, and TBil were all elevated in the APAP experimental group. The levels of ALT (P = 0.004), AST (P = 0.002), and TBil (P = 0.012) were significantly elevated among the mutant group compared to those in the wild-type group, and the expression levels of these indicators were also significantly elevated among the homozygous mutant group compared to those in the heterozygous mutant group (P = 0.003, 0 and 0.006). In addition, the ALP levels were higher in the heterozygous mutation group than those in the homozygous mutant group (P = 0.085) and wild-type group mice, but the difference was only statistically significant compared to wild-type mice (P = 0.002). HE staining results showed that mice in the APAP experimental group had hepatocyte degeneration, necrosis, and increased inflammatory cell infiltration, which was mostly evident in mutant mice. Simultaneously, the F4/80 immunohistochemical staining results showed that brown granules were visible in the liver tissue of APAP experimental group mice, and its expression levels were significantly enhanced compared to the blank control group. Conclusion: APAP-induced liver function abnormalities were associated with the ALDH2 gene polymorphism. The liver injury symptoms were increased in ALDH2 mutant mice following APAP modeling, and the ALDH2 gene defect may alleviate, to some extent, APAP-induced liver function abnormalities.

Selenoprotein H mediates low selenium-related cognitive decline through impaired oligodendrocyte myelination with disrupted hippocampal lipid metabolism in female mice.

Low selenium levels are closely associated with reduced cognitive performance and lipid dysregulation, yet the mechanism of action remains unclear. The physiological function of selenium is primarily mediated by selenoproteins. Selenoprotein H (SELENOH), as one of the selenium-containing proteins, has an unelucidated role in regulating cognitive status and lipid metabolism. In this study, we established a Selenoh gene knockout (HKO) mouse model to investigate whether Selenoh mediates the impact of selenium on cognitive function. We found that HKO mice showed a significant decline in cognition compared with the wild-type (HWT) littermates, and were not affected by deficient or excessive selenium, while no differences in anxiety and depression behavior were observed. HKO mice showed reduced myelin basic protein expression in hippocampal oligodendrocytes, with decreased glycolipid levels and increased phospholipid and sphingolipid levels in the hippocampus. Furthermore, the high-fat diet (HFD) exerted no effect on cognition and limited impact on the gene profile in the hippocampus of HKO mice. Compared with those of HWT mice, the myelination pathways in the hippocampus of HKO mice were downregulated as revealed by RNA-seq, which was further confirmed by the reduced expression levels of myelin-related proteins. Finally, HKO increased the expression of hippocampal fatty acid transporter (FATP) 4, and HFD increased the FATP4 expression in HWT mice but not in HKO mice. In summary, our study demonstrated that HKO induced cognitive decline by impairing myelination in oligodendrocytes with disrupted hippocampal lipid metabolism, which provided a novel viewpoint on the selenoprotein-mediated neurodegenerative diseases of selenium.

Cav3.2 channel regulates cerebral ischemia/reperfusion injury: a promising target for intervention.

JOURNAL/nrgr/04.03/01300535-202419110-00028/figure1/v/2024-03-08T184507Z/r/image-tiff Calcium influx into neurons triggers neuronal death during cerebral ischemia/reperfusion injury. Various calcium channels are involved in cerebral ischemia/reperfusion injury. Cav3.2 channel is a main subtype of T-type calcium channels. T-type calcium channel blockers, such as pimozide and mibefradil, have been shown to prevent cerebral ischemia/reperfusion injury-induced brain injury. However, the role of Cav3.2 channels in cerebral ischemia/reperfusion injury remains unclear. Here, in vitro and in vivo models of cerebral ischemia/reperfusion injury were established using middle cerebral artery occlusion in mice and high glucose hypoxia/reoxygenation exposure in primary hippocampal neurons. The results showed that Cav3.2 expression was significantly upregulated in injured hippocampal tissue and primary hippocampal neurons. We further established a Cav3.2 gene-knockout mouse model of cerebral ischemia/reperfusion injury. Cav3.2 knockout markedly reduced infarct volume and brain water content, and alleviated neurological dysfunction after cerebral ischemia/reperfusion injury. Additionally, Cav3.2 knockout attenuated cerebral ischemia/reperfusion injury-induced oxidative stress, inflammatory response, and neuronal apoptosis. In the hippocampus of Cav3.2-knockout mice, calcineurin overexpression offset the beneficial effect of Cav3.2 knockout after cerebral ischemia/reperfusion injury. These findings suggest that the neuroprotective function of Cav3.2 knockout is mediated by calcineurin/nuclear factor of activated T cells 3 signaling. Findings from this study suggest that Cav3.2 could be a promising target for treatment of cerebral ischemia/reperfusion injury.

PDCD4 deficiency improved 4-vinylcyclohexene dioxide-induced mouse premature ovarian insufficiency

Research questionWhat role does programmed cell death 4 (PDCD4) play in premature ovarian insufficiency (POI)? DesignA PDCD4 gene knockout (PDCD4–/–) mouse model was constructed, a POI mouse model was established similar to human POI with 4-vinylcyclohexene dioxide (VCD), a PDCD4-overexpressed adenovirus was designed and the regulatory role in POI in vitro and in vivo was investigated. ResultsPDCD4 expression was significantly increased in the ovarian granulosa cells of patients with POI (P ≤ 0.002 protein and mRNA) and mice with VCD-induced POI (P < 0.001 protein expression in both mouse ovaries and granulosa cells). In POI-induced mice model, PDCD4 knockouts significantly increased anti-Müllerian hormone, oestrodiol and numbers of developing follicles, and the PI3K-AKT-Bcl2/Bax signalling pathway is involved in it. ConclusionThe expression and regulation of PDCD4 significantly affects the POI pathology in a mouse model. This effect is closely related to the regulation of Bcl2/Bax and the activation of the PI3K-AKT signalling pathway.

Neuropeptide Y Attenuates Aging-Induced Bone Marrow Niche Defects and Rejuvenate Aged Hematopoietic Stem/Progenitor Cells

Aging-associated defects in hematopoiesis are linked to declining in hematopoietic stem cell (HSC) self-renewal and multilineage differentiation capacity. The bone marrow (BM) microenvironment/niche has been suggested to influence HSC aging. However, the factors/mechanisms whereby the BM niche mediates hematopoietic stem and progenitor (HSPC) aging are poorly understood. We recently discovered that neuropeptide Y (NPY) plays a crucial role in HSPC trafficking by regulating the BM vascular gateway function (PMCID: PMC5707149). NPY is one of the most abundant neurotransmitters released in the BM by sympathetic nerve fibers. In this study, we found that the aging-induced deficit of NPY levels in the BM plays a critical role in niche defects and HSPC aging by impairing the molecular pathways involved in regulating oxidative stress, senescence, and stemness. The measurement of NPY levels in the BM of young (3-4 months) and aged (18-23 months) mice showed lower NPY levels in aged mice than in young mice (2.2-fold), which was associated with a decrease in mesenchymal stem cells (MSC) (2-fold, p=0.008) and endothelial cells (EC) (1.5-fold, p&amp;lt;0.05) numbers in the BM. In addition, we found that circulating NPY levels decline in the older human population (65-83 years) than in young/ middle age (23-55 years) individuals (1.5-fold, p=0.01). We hypothesized if aging-induced NPY deficit contributes to BM niche defects and HSPC aging, then NPY supplementation may attenuate/revert these processes. To explore this possibility, we administered exogenous NPY in aged mice (100ng/mouse) for 15 consecutive days and evaluated HSPC and niche cells. NPY supplementation in aged mice (18-23 months) substantially reverted aged HSPC phenotype, including NPY decreased the accumulation of HSC-enriched SLAM LSK (Lin-Sca-1+c-Kit+CD48-CD150+) in the BM (1.8-fold, p&amp;lt;0.05) and increased the HPC clonal expansion (1.9-fold, p&amp;lt;0.05) measured by flow cytometry and CFU-C assay, respectively. Competitive BM transplantation studies using donor cells from young, aged, or NPY-treated aged mice into lethally irradiated syngeneic mice showed myeloid biased repopulation from aged mice donor cells than young mice donor cells, and that was attenuated in NPY-treated aged mice donor cells (33%, p=0.003). In addition, NPY substantially reverted aging-related reduction in BM MSC (CD45-Ter119-CD31-Nestin+PDGFR+CD51+) and EC (CD45-Ter119-CD31+VE-cadherin+) numbers, and BM MSC clonal expansion (2-fold, p&amp;lt;0.05) as measured by CFU-F assay. To further validate NPY's role in the aged BM niche and HSPC function, we used NPY gene overexpressing and knockout (KO) mouse models. Compared to wild-type (WT) aged mice, NPY overexpressing aged mice showed a substantial increase in BM niche MSC numbers (1.4-fold, P=0.03) and improved HSPC clonal expansion (2.5-fold, P=0.002). In contrast, 16-18 months old NPY KO mice showed enhanced aging than aged WT mice, reflected by fewer MSC counts and lower HSPC clonal expansion (MSC: 1.6-fold, and CFU-F: 1.5-fold, &amp;lt;0.05). Oxidative stress and senescence are the hallmarks of aging. To examine if NPY attenuates HSPC aging by controlling oxidative and senescence, we measured reactive oxygen species (ROS) and senescence in SLAM-LSK of young, aged, and NPY-treated aged mice using CellROX and anti-p16 antibodies, respectively. The SLAM-LSK from aged mice showed higher expression of ROS (9-fold, p=0.03) and p16 than young mice (1.7-fold). However, ROS and p16 expression were decreased in NPY-treated aged mice (ROS: 2-fold, and p16: 1.3-fold, p&amp;lt;0.05). To identify the molecular pathways involved in NPY-mediated attenuation of HSPC aging, we performed RNA sequencing analysis of SLAM-LSK cells. Aged mice SLAM LSK showed impaired expression of genes involved in pathways regulating antioxidant defense, DNA damage repair and stemness than in young mice SLAM LSK. However, NPY treatment in aged mice substantially restored these pathways in SLAM LSK. In conclusion, our studies suggest that an aging-induced deficit in NPY levels in the BM is a crucial lesion contributing to niche defects and HSPC aging. Thus, NPY supplementation or protection of NPY-producing nerve fibers in the BM can attenuate/restore aging-induced niche damage and rejuvenate aged HSPC.

Fast, multiplexable and efficient somatic gene deletions in adult mouse skeletal muscle fibers using AAV-CRISPR/Cas9

Molecular screens comparing different disease states to identify candidate genes rely on the availability of fast, reliable and multiplexable systems to interrogate genes of interest. CRISPR/Cas9-based reverse genetics is a promising method to eventually achieve this. However, such methods are sorely lacking for multi-nucleated muscle fibers, since highly efficient nuclei editing is a requisite to robustly inactive candidate genes. Here, we couple Cre-mediated skeletal muscle fiber-specific Cas9 expression with myotropic adeno-associated virus-mediated sgRNA delivery to establish a system for highly effective somatic gene deletions in mice. Using well-characterized genes, we show that local or systemic inactivation of these genes copy the phenotype of traditional gene-knockout mouse models. Thus, this proof-of-principle study establishes a method to unravel the function of individual genes or entire signaling pathways in adult skeletal muscle fibers without the cumbersome requirement of generating knockout mice.

Abstract P2170: Rescue Of Ctni Null Mice By Application Of Aav-9 Encoding Ctni Gene

Background: Heart diastolic dysfunction can be caused by cardiac troponin I (cTnI) deficiencies or mutations. Our lab has created a unique cTnI gene knockout mouse model, demonstrating that homozygous cTnI null mice (-/-) die at postnatal day 18 (p18). In this study, we have applied gene therapy using adeno-associated virus serotype 9 (AAV9) carrying wild-type cTnI gene to rescue mice from a lethal cTnI knockout phenotype. Methods: The proposed gene therapy strategies involve novel approaches delivering AAV9 via intraperitoneal injection in newborn cTnI mice (-/-), employing a cardiac-specific promoter (chicken cTnT promoter) to express cTnI protein in myocardial cells. Results: Mice treated with doses ranging from 2e10 10 vg/g (vector genome per gram) to 8e10 10 vg/g at p10 displayed an increased life span compared to mice without treatment. 5e10 10 vg/g multi-injection two-dose treatment at p10 and p14 demonstrated 22 additional days of life past p18 ( Figure A) . Using echocardiography, treated mice showed significant improvement in left ventricular diastolic function compared to mice without treatment. In addition, western blotting of cardiac samples demonstrated restored cTnI proteins in treated mice. Conclusion: The in vivo gene therapy approach shows that our mouse model can survive 2-3 weeks longer than untreated cTnI null mice. These data suggest AAV-9 serotype with a cardiac-specific promoter is a promising tool in treating cardiomyopathies by restoring cardiac proteins and reversing diastolic dysfunction.

Periodontal ligament-associated protein-1 knockout mice regulate the differentiation of osteoclasts and osteoblasts through TGF-β1/Smad signaling pathway.

It has been known that periodontal ligament-associated protein-1 (PLAP-1/Asporin) not only inhibits cartilage formation in osteoarthritis, but it also influences the healing of skull defect. However, the effect and mechanism of PLAP-1/Asporin on the mutual regulation of osteoclasts and osteoblasts in periodontitis are not clear. In this study, we utilized a PLAP-1/Asporin gene knockout (KO) mouse model to research this unknown issue. We cultured mouse bone marrow mesenchymal stem cells with Porphyromonas gingivalis lipopolysaccharide (P.g. LPS) for osteogenic induction in vitro. The molecular mechanism of PLAP-1/Asporin in the regulation of osteoblasts was detected by immunoprecipitation, immunofluorescence, and inhibitors of signaling pathways. The results showed that the KO of PLAP-1/Asporin promoted osteogenic differentiation through transforming growth factor beta 1 (TGF-β1)/Smad3 in inflammatory environments. We further found the KO of PLAP-1/Asporin inhibited osteoclast differentiation and promoted osteogenic differentiation through the TGF-β1/Smad signaling pathway in an inflammatory coculture system. The experimental periodontitis model was established by silk ligation and the alveolar bone formation in PLAP-1/Asporin KO mice was promoted through TGF-β1/Smad3 signaling pathway. The subcutaneous osteogenesis model in nude mice also confirmed that the KO of PLAP-1/Asporin promoted bone formation by the histochemical staining. In conclusion, PLAP-1/Asporin regulated the differentiation of osteoclasts and osteoblasts through TGF-β1/Smad signaling pathway. The results of this study lay a theoretical foundation for the further study of the pathological mechanism underlying alveolar bone resorption, and the prevention and treatment of periodontitis.

Construction of a Brain-specific SLC23A2 Gene Knockout Mice Model

Vitamin C (VC) is a key antioxidant of the Central Nervous System (CNS) and SLC23A2 (SVCT2) is the only transporter that actively transports VC into the brain. While the existing animal models of VC deficiency are in the whole body, the essential role of VC in brain development remains elusive. In our study presented here, the CRISPR/Cas9 technology was applied for the construction of a C57BL/6J-SLC23A2 em1(flox)Smoc mouse model, which was crossed with the Glial fibrillary acidic protein-driven Cre Recombinase (GFAP-Cre) genotype mice to generate a conditional knockout model of SLC23A2(SVCT2) gene in mice brain (GFAP-Cre;SLC23A2 flox/flox) after generations of crossbreeding. Our results showed that the expression of SVCT2 in GFAP-Cre;SLC23A2 flox/flox (Cre;svct2 f/f) mice brain was significantly decreased, and consistently, the expression of Neuronal nuclei antigen (NeuN), Glial fibrillary acidic protein (GFAP), calbindin-28k, brain-derived neurotrophic factor (BDNF) was down-regulated but Ionized calcium binding adapter molecule 1 (Iba-1) was up-regulated in Cre;svct2 f/f mice brain tissues. On the other hand, the levels of Glutathione, Reduced (GSH), myeloperoxidase (MDA), 8-isoprostane, tumor necrosis factor-α (TNF-α) and interleukin-6(IL-6) were significantly increased, but the levels of VC in brain tissue of the model group were decreased in Cre;svct2 f/f mice brain tissues, indicating the protective effect of VC against oxidative stress and inflammation during pregnancy. Thus, the conditional knockout of the SLC23A2 gene in the brain of mouse was successfully established by the CRISPR/Cas9 technology in our study, providing an effective animal model for studying the role of VC in fetal brain development.

FBLN2 Abates Epigenetic-mediated Salt-sensitive Hypertension Through Regulation of NCC Trafficking

Epigenetics play a role in the development of hypertension. However, the precise mechanisms of how histone modification in renal tubule cells regulates blood pressure is largely undiscovered. In this study, we investigated the role of renal Kdm6a in salt-sensitive hypertension using a kidney tubular cell-specific Kdm6a gene knockout (Kdm6a cKO) mouse model. First, we demonstrated that knockout of Kdm6a in the renal tubular cells resulted in an increase of H3K27me3 associated with the development of hypertension in both male and female Kdm6a cKO mice. To explore the mechanism of H3K27me3-mediated hypertension, we performed a ChIP seq to identify the target genes of H3K27me3 in the renal tubular cells. Data showed that H3K27me3 regulated several physiological pathways in the renal tubular cells, including sodium transport in proximal convoluted tubule (PCT), and blood pressure regulation in distal convoluted tubule (DCT). Indeed, we found that the expressions of NCC, NKCC2, and AQP2 were upregulated in kidneys of Kdm6a cKO mice. Furthermore, ChIP seq data reveals that FBLN2, an extracellular matrix protein, appears to be a target of H3K27me3, which was downregulated in the kidney of Kdm6a cKO mice determined by real-time PCR, Western, and IHC. Notably, we demonstrated that FBLN2 abated high salt-induced hypertension through downregulation of NCC, NKCC2, and AQP2 in the tubular cells of Kdm6a cKO mice. Interestingly, pre-treatment of FBLN2 prevented salt-sensitive hypertension in these mice. Additionally, we have provided evidence that FBLN2 may regulate blood pressure through mediating the NCC cell membrane trafficking. Using a super high-resolution microscopy, we demonstrated that FBLN2 reduced levels of cell surface NCC in DCT cells cultured in hypotonic medium. Here, we have first demonstrated that FBLN2 may be a target gene of H3K27me3 in epigenetic-mediated hypertension. FBLN2 treatment may prevent and treat salt-sensitive hypertension. NIH This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Cholesterol-Amino-Phosphate (CAP) Derived Lipid Nanoparticles for Delivery of Self-Amplifying RNA and Restoration of Spermatogenesis in Infertile Mice.

Male infertility caused by genetic mutations is an important type of infertility. Currently, there is no reliable method in the clinic to address this medical need. The emergence of mRNA therapy provides a possible strategy for restoring mutant genes in the reproductive system. However, effective delivery of mRNA to spermatocytes remains a formidable challenge. Here a series of cholesterol-amino-phosphate (CAP) lipids are reported by integrating three bioactive moieties into a geometric structure, which is favorable for mRNA delivery. The results demonstrate that CAP-derived lipid nanoparticles (CAP LNPs) can deliver RNA including traditional mRNA and self-amplifying RNA (saRNA) encoding DNA Meiotic Recombinase 1 (Dmc1) protein in spermatocytes and treat male infertility caused by the Dmc1 gene mutation. Notably, the delivery efficiency of CAP LNPs is significantly higher than that of the MC3 and ALC-0315 LNPs, which is consistent with the design of CAP molecules. More importantly, a single injection of CAP LNPs-saRNA can produce Dmc1 protein for an extended period, which restores the spermatogenesis in the Dmc1 gene knockout mouse model. Overall, this study proves the concept of LNPs for the delivery of mRNA to spermatocytes, which provides a unique method to probe male infertility caused by the genetic mutation.