Generation Of Conditional Knockout Mice Research Articles

Floxed Il1rl2 Locus with mCherry Reporter Element Reveals Distinct Expression Patterns of the IL-36 Receptor in Barrier Tissues.

IL-36 cytokines are emerging as beneficial in immunity against pathogens and cancers but can also be detrimental when dysregulated in autoimmune and autoinflammatory conditions. Interest in targeting IL-36 activity for therapeutic purposes is rapidly growing, yet many unknowns about the functions of these cytokines remain. Thus, the availability of robust research tools is essential for both fundamental basic science and pre-clinical studies to fully access outcomes of any manipulation of the system. For this purpose, a floxed Il1rl2, the gene encoding the IL-36 receptor, mouse strain was developed to facilitate the generation of conditional knockout mice. The targeted locus was engineered to contain an inverted mCherry reporter sequence that upon Cre-mediated recombination will be flipped and expressed under the control of the endogenous Il1rl2 promoter. This feature can be used to confirm knockout in individual cells but also as a reporter to determine which cells express the IL-36 receptor IL-1RL2. The locus was confirmed to function as intended and further used to demonstrate the expression of IL-1RL2 in barrier tissues. Il1rl2 expression was detected in leukocytes in all barrier tissues. Interestingly, strong expression was observed in epithelial cells at locations in direct contact with the environment such as the skin, oral mucosa, the esophagus, and the upper airways, but almost absent from epithelial cells at more inward facing sites, including lung alveoli, the small intestine, and the colon. These findings suggest specialized functions of IL-1RL2 in outward facing epithelial tissues and cells. The generated mouse model should prove valuable in defining such functions and may also facilitate basic and translational research.

Generation and characterization of cerebellar granule neurons specific knockout mice of Golli-MBP.

Golli-myelin basic proteins, encoded by the myelin basic protein gene, are widely expressed in neurons and oligodendrocytes in the central nervous system. Further, prior research has shown that Golli-myelin basic protein is necessary for myelination and neuronal maturation during central nervous system development. In this study, we established Golli-myelin basic protein-floxed mice to elucidate the cell-type-specific effects of Golli-myelin basic protein knockout through the generation of conditional knockout mice (Golli-myelin basic proteinsfl/fl; E3CreN), in which Golli-myelin basic proteins were specifically deleted in cerebellar granule neurons, where Golli-myelin basic proteins are expressed abundantly in wild-type mice. To investigate the role of Golli-myelin basic proteins in cerebellar granule neurons, we further performed histopathological analyses of these mice, with results indicating no morphological changes or degeneration of the major cellular components of the cerebellum. Furthermore, behavioral analysis showed that Golli-myelin basic proteinsfl/fl; E3CreN mice were healthy and did not display any abnormal behavior. These results suggest that the loss of Golli-myelin basic proteins in cerebellar granule neurons does not lead to cerebellar perturbations or behavioral abnormalities. This mouse model could therefore be employed to analyze the effect of Golli-myelin basic protein deletion in specific cell types of the central nervous system, such as other neuronal cells and oligodendrocytes, or in lymphocytes of the immune system.

Evidence for a Role of 5-HT-glutamate Co-releasing Neurons in Acute Stress Mechanisms.

A major subpopulation of midbrain 5-hydroxytryptamine (5-HT) neurons expresses the vesicular glutamate transporter 3 (VGLUT3) and co-releases 5-HT and glutamate, but the function of this co-release is unclear. Given the strong links between 5-HT and uncontrollable stress, we used a combination of c-Fos immunohistochemistry and conditional gene knockout mice to test the hypothesis that glutamate co-releasing 5-HT neurons are activated by stress and involved in stress coping. Acute, uncontrollable swim stress increased c-Fos immunoreactivity in neurons co-expressing VGLUT3 and the 5-HT marker tryptophan hydroxylase 2 (TPH2) in the dorsal raphe nucleus (DRN). This effect was localized in the ventral DRN subregion and prevented by the antidepressant fluoxetine. In contrast, a more controllable stressor, acute social defeat, had no effect on c-Fos immunoreactivity in VGLUT3-TPH2 co-expressing neurons in the DRN. To test whether activation of glutamate co-releasing 5-HT neurons was causally linked to stress coping, mice with a specific deletion of VGLUT3 in 5-HT neurons were exposed to acute swim stress. Compared to wildtype controls, the mutant mice showed increased climbing behavior, a measure of active coping. Wildtype mice also showed increased climbing when administered fluoxetine, revealing an interesting parallel between the behavioral effects of genetic loss of VGLUT3 in 5-HT neurons and 5-HT reuptake inhibition. We conclude that 5-HT-glutamate co-releasing neurons are recruited by exposure to uncontrollable stress. Furthermore, natural variation in the balance of 5-HT and glutamate co-released at the 5-HT synapse may impact stress susceptibility.

Gene knockout of RNA binding motif 5 in the brain alters RIMS2 protein homeostasis in the cerebellum and Hippocampus and exacerbates behavioral deficits after a TBI in mice

RNA binding motif 5 (RBM5) is a tumor suppressor in cancer but its role in the brain is unclear. We used conditional gene knockout (KO) mice to test if RBM5 inhibition in the brain affects chronic cortical brain tissue survival or function after a controlled cortical impact (CCI) traumatic brain injury (TBI). RBM5 KO decreased baseline contralateral hemispheric volume (p < 0.0001) and exacerbated ipsilateral tissue loss at 21 d after CCI in male mice vs. wild type (WT) (p = 0.0019). CCI injury, but not RBM5 KO, impaired beam balance performance (0-5d post-injury) and swim speed on the Morris Water Maze (MWM) (19-20d) (p < 0.0001). RBM5 KO was associated with mild learning impairment in female mice (p = 0.0426), reflected as a modest increase in escape latency early in training (14-18d post-injury). However, KO did not affect spatial memory at 19d post-injury in male or in female mice but it was impaired by CCI in females (p = 0.0061). RBM5 KO was associated with impaired visual function in male mice on the visible platform test at 20d post-injury (p = 0.0256). To explore signaling disturbances in KOs related to behavior, we first cross-referenced known brain-specific RBM5-regulated gene targets with genes in the curated RetNet database that impact vision. We then performed a secondary literature search on RBM5-regulated genes with a putative role in hippocampal function. Regulating synaptic membrane exocytosis 2 (RIMS) 2 was identified as a gene of interest because it regulates both vision and hippocampal function. Immunoprecipitation and western blot confirmed protein expression of a novel ~170 kDa RIMS2 variant in the cerebellum, and in the hippocampus, it was significantly increased in KO vs WT (p < 0.0001), and in a sex-dependent manner (p = 0.0390). Furthermore, male KOs had decreased total canonical RIMS2 levels in the cerebellum (p = 0.0027) and hippocampus (p < 0.0001), whereas female KOs had increased total RIMS1 levels in the cerebellum (p = 0.0389). In summary, RBM5 modulates brain function in mammals. Future work is needed to test if RBM5 dependent regulation of RIMS2 splicing effects vision and cognition, and to verify potential sex differences on behavior in a larger cohort of mice.

AAV-based gene therapy prevents and halts the progression of dilated cardiomyopathy in a mouse model of phosphoglucomutase 1 deficiency (PGM1-CDG)

Phosphoglucomutase 1 (PGM1) deficiency is recognized as the third most common N-linked congenital disorders of glycosylation (CDG) in humans. Affected individuals present with liver, musculoskeletal, endocrine, and coagulation symptoms; however, the most life-threatening complication is the early onset of dilated cardiomyopathy (DCM). Recently, we discovered that oral D-galactose supplementation improved liver disease, endocrine, and coagulation abnormalities, but does not alleviate the fatal cardiomyopathy and the associated myopathy. Here we report on left ventricular ejection fraction (LVEF) in 6 individuals with PGM1-CDG. LVEF was pathologically low in most of these individuals and varied between 10% and 65%. To study the pathobiology of the cardiac disease observed in PGM1-CDG, we constructed a novel cardiomyocyte-specific conditional Pgm2 gene (mouse ortholog of human PGM1) knockout (Pgm2 cKO) mouse model. Echocardiography studies corroborated a DCM phenotype with significantly reduced ejection fraction and left ventricular dilation similar to those seen in individuals with PGM1-CDG. Histological studies demonstrated excess glycogen accumulation and fibrosis, while ultrastructural analysis revealed Z-disk disarray and swollen/fragmented mitochondria, which was similar to the ultrastructural pathology in the cardiac explant of an individual with PGM1-CDG. In addition, we found decreased mitochondrial function in the heart of KO mice. Transcriptomic analysis of hearts from mutant mice demonstrated a gene signature of DCM. Although proteomics revealed only mild changes in global protein expression in left ventricular tissue of mutant mice, a glycoproteomic analysis unveiled broad glycosylation changes with significant alterations in sarcolemmal proteins including different subunits of laminin-211, which was confirmed by immunoblot analyses. Finally, augmentation of PGM1 in KO mice via AAV9-PGM1 gene replacement therapy prevented and halted the progression of the DCM phenotype.

Constitutive and conditional gene knockout mice for the study of intervertebral disc degeneration: Current status, decision considerations, and future possibilities.

There have been an increasing number of patients with degenerative disc diseases due to the aging population. In light of this, studies on the pathogenesis of intervertebral disc degeneration have become a hot topic, and gene knockout mice have become a valuable tool in this field of research. With the development of science and technology, constitutive gene knockout mice can be constructed using homologous recombination, zinc finger nuclease, transcription activator-like effector nuclease technology and clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9) system, and conditional gene knockout mice can be constructed using the Cre/LoxP system. The gene-edited mice using these techniques have been widely used in the studies on disc degeneration. This paper reviews the development process and principles of these technologies, functions of the edited genes in disc degeneration, advantages, and disadvantages of different methods and possible targets of the specific Cre recombinase in intervertebral discs. Recommendations for the choice of suitable gene-edited model mice are presented. At the same time, possible technological improvements in the future are also discussed.

Generation of Floxed Mice by Sequential Electroporation.

Generation of conditional knockout mice using the Cre-loxP system is essential for the analysis of gene functions. The use of CRISPR-Cas9 in combination with two sets of guide RNAs and single-stranded oligonucleotides including loxP sites enables simultaneous insertion of two loxP sequences. Unfortunately, this method induces double-strand breaks at two sites in the same chromosome, which causes an undesirable large chromosomal deletion and reduces the flanked loxP (flox) rate. To overcome this problem, we have developed a method that sequentially introduces each loxP sequence by electroporation at the one- and two-cell embryonic stages, respectively. This sequential electroporation method improves the floxing efficiency compared with the conventional simultaneous method, leading to a high yield of offspring with floxed alleles.

Analysis of the Function of CCN2 in Tubular Epithelium Cells with a Focus on Renal Fibrogenesis.

Renal interstitial fibrosis is the final common pathway in the process of all kidney diseases, and it results in chronic kidney disease. CCN2 is an important factor in the pathogenesis of renal interstitial fibrosis, and analysis of its function can lead to treatments for chronic kidney disease. Since CCN2 knockout mice are developmentally lethal, generation of conditional knockout mice is essential for in vivo analysis. Since CCN2 is expressed in a variety of cells in the kidney, including podocytes, mesangial cells, pericytes, and tubular epithelial cells, it is necessary to perform cell-specific verification of the cells that play a central role in fibrosis. However, cell-specific validation using the Cre/loxP system in vivo has only been performed in mesangial cells. In our research program, we are focusing on the role of CCN2 in tubular epithelial cells in renal fibrogenesis. In this report, we introduce the creation of a tubular epithelial cell-specific knockout model and method of its analysis.

Astrocyte-derived hepcidin controls iron traffic at the blood-brain-barrier via regulating ferroportin 1 of microvascular endothelial cells

Brain iron dysregulation associated with aging is closely related to motor and cognitive impairments in neurodegenerative diseases. The regulation of iron traffic at the blood-brain barrier (BBB) is crucial to maintain brain iron homeostasis. However, the specific mechanism has not been clarified in detail. Using various conditional gene knockout and overexpression mice, as well as cell co-culture of astrocyte and bEND.3 in the transwell, we found that astrocyte hepcidin knockdown increased the expression of ferroportin 1 (FPN1) of brain microvascular endothelial cells (BMVECs), and that it also induced brain iron overload and cognitive decline in mice. Moreover, BMVECs FPN1 knockout decreased iron contents in the cortex and hippocampus. Furthermore, hepcidin regulates the level of FPN1 of BMVECs with conditional gene overexpression in vivo and in vitro. Our results revealed that astrocytes responded to the intracellular high iron level and increased the secretion of hepcidin, which in turn diminished iron uptake at BBB from circulation through directly regulating FPN1 of BMVECs. Our results demonstrate that FPN1 of BMVECs is a gateway for iron transport into the brain from circulation, and the controller of this gateway is hepcidin secreted by astrocyte at its endfeet through physical contact with BMVECs. This regulation is indeed the major checkpoint for iron transport from the blood circulation to the brain. This study delineates the pathway and regulation of iron entry into the brain, providing potential therapeutic targets for iron dysregulation-related neurological diseases.

Osteocytes regulate bone anabolic response to mechanical loading in male mice via activation of integrin α5

Physical mechanical stimulation can maintain and even increase bone mass. Here, we report an important role of osteocytic integrin α5 in regulating the anabolic response of bone to mechanical loading using an Itga5 conditional gene knockout (cKO) mouse model. Integrin α5 gene deletion increased apoptotic osteocytes and reduced cortical anabolic responses to tibial compression including decreased endosteal osteoblasts and bone formation, and increased endosteal osteoclasts and bone resorption, contributing to the decreased bone area fraction and biomechanical properties, leading to an enlarged bone marrow area in cKO mice. Similar disruption of anabolic responses to mechanical loading was also detected in cKO trabecular bone. Moreover, integrin α5 deficiency impeded load-induced Cx43 hemichannel opening, and production and release of PGE2, an anabolic factor, resulting in attenuated effects of the loading on catabolic sclerostin (SOST) reduction and anabolic β-catenin increase. Together, this study shows an indispensable role of integrin α5 in osteocytes in the anabolic action of mechanical loading on skeletal tissue through activation of hemichannels and PGE2-evoked gene expression. Integrin α5 could act as a potential new therapeutic target for bone loss, especially in the elderly population with impeded mechanical sensitivity.

Intestinal Claudin-7 deficiency impacts the intestinal microbiota in mice with colitis

BackgroundIntestinal epithelial cells form a physical barrier that protects the intestine against the intestinal microbiota through tight junctions (TJs) and adhesive junctions, while barrier disruption may lead to inflammatory bowel disease (IBD). Claudin-7 (Cldn7) has been implicated in this protection as an important member of TJs. Here, we experimentally study the effect of Cldn7 deletion on intestinal microbiota in colitis.MethodsColitis model was established based on inducible intestinal conditional Cldn7 gene knockout mice (Cldn7fl/fl; villin-CreERT2), by feeding with dextran sodium sulfate (DSS). AB-PAS staining and immunohistochemical staining of Muc2 mucin were used to detect the effect of Cldn7 deficiency on the mucus layer of mice with colitis, and fluorescence in situ hybridization was used to detect how Cldn7 promotes spatial separation of the gut microbiota from the host. The microbiota population was characterized by high-throughput 16S rRNA gene sequencing of DNA extracted from fecal samples.ResultsCompared with the controls, Cldn7 knockout increased susceptibility to colitis, including greater degree of weight loss, colon shortening, and a significantly higher disease activity index score. DSS-treated Cldn7 knockout mice promoted the migration of bacteria to the intestinal epithelium to some extent by damaging the intestinal mucus layer. Sequencing of 16S rRNA showed that DSS-treated Cldn7 knockout mice reduced the gut microbiota diversity and had greater relative abundance of Escherichia coli. LEfSe analysis indicated that Escherichia coli may be the key bacteria in Cldn7 knockout mice during DSS-induced colitis. Furthermore, the Tax4Fun analysis predicted that DSS-treated Cldn7 knockout mice enriched for microbiota impacting infectious diseases, immune system and metabolic functions.ConclusionsOur data suggests an association between intestinal Cldn7 knockout and microbiota dysbiosis during inflammatory events.

High-Survival Rate After Microinjection of Mouse Oocytes and Early Embryos With mRNA by Combining a Tip Pipette and Piezoelectric-Assisted Micromanipulator.

Utilizing microinjection to introduce biological molecules such as DNA, mRNA, siRNA, and proteins into the cell is well established to study oocyte maturation and early embryo development in vitro. However, microinjection is an empirical technology. The cellular survival after microinjection is mainly dependent on the operator, and an experienced operator should be trained for a long time, from several months to years. Optimizing the microinjection to be highly efficient and quickly learned should be helpful for new operators and some newly established laboratories. Here, we combined the tip pipette and piezo-assisted micromanipulator to microinject the oocyte and early embryos at different stages of mouse. The results showed that the survival rate after microinjection was more than 85% for cumulus–oocyte complex, germinal vesicle oocyte, two-cell, and four-cell embryos, and close to 100% for MII oocyte and zygotes. The high-rate survival of microinjection can save many experimental samples. Thus, it should be helpful in studying some rare animal models such as aging and conditional gene knockout mice. Furthermore, our protocol is much easier to learn for new operators, who can usually master the method proficiently after several training times. Therefore, we would like to publicly share this experience, which will help some novices master microinjection skillfully and save many laboratory animals.

Identification of Jmjd3 as an Essential Epigenetic Regulator of Hox Gene Temporal Collinear Activation for Body Axial Patterning in Mice.

Body axial patterning develops via a rostral-to-caudal sequence and relies on the temporal colinear activation of Hox genes. However, the underlying mechanism of Hox gene temporal colinear activation remains largely elusive. Here, with small-molecule inhibitors and conditional gene knockout mice, we identified Jmjd3, a subunit of TrxG, as an essential regulator of temporal colinear activation of Hox genes with its H3K27me3 demethylase activity. We demonstrated that Jmjd3 not only initiates but also maintains the temporal collinear expression of Hox genes. However, we detected no antagonistic roles between Jmjd3 and Ezh2, a core subunit of PcG repressive complex 2, during the processes of axial skeletal patterning. Our findings provide new insights into the regulation of Hox gene temporal collinear activation for body axial patterning in mice.

Claudin-7 deficiency promotes stemness properties in colorectal cancer through Sox9-mediated Wnt/\u03b2-catenin signalling

BackgroundColorectal cancer (CRC) is a common malignant tumour of the digestive tract that is characterized by high patient morbidity and mortality rates. Claudin-7 (Cldn7), a tight junction protein, was recently reported to function as a candidate tumour suppressor gene in CRC. Our previous study demonstrated that the large intestine of C57/BL6 mice showed intestinal adenomas and abnormal Ki67 expression and distribution in the intestinal crypt when Cldn7 was knocked out. The aim of this study was to further investigate whether Cldn7 deficiency has non-tight junction functions, affects intestinal stemness properties, promotes CRC and to determine the specific mechanism.MethodsCell proliferation assays, migration assays, apoptosis assays, tumour sphere formation assays in vitro, and subcutaneous xenograft models in vivo were used to determine the effects of Cldn7 knockdown on the biological characteristics of CRC stem cells. Western blotting, qPCR and immunofluorescence staining were performed to identify the epithelial-mesenchymal transition and the activation of Wnt/β-catenin pathway in CRC stem cells. Cldn7 inducible conditional gene knockout mice and immunohistochemical staining further verified this hypothesis in vivo. The mechanism and target of Cldn7 were determined by performing a chromatin immunoprecipitation (ChIP) assay and coimmunoprecipitation (CoIP) assay.ResultsCldn7 knock down in CRC stem cells promoted cell proliferation, migration, and globular growth in serum-free medium and the ability to form xenograft tumours; cell apoptosis was inhibited, while the cellular epithelial-mesenchymal transition was also observed. These changes in cell characteristics were achieved by activating the Wnt/β-catenin pathway and promoting the expression of downstream target genes after β-catenin entry into the nucleus, as observed in CRC cell lines and Cldn7 gene knockout mouse experiments. Using ChIP and CoIP experiments, we initially found that Cldn7 and Sox9 interacted at the protein level to activate the Wnt/β-catenin pathway.ConclusionsBased on our research, Cldn7 deficiency confers stemness properties in CRC through Sox9-mediated Wnt/β-catenin signalling. This result clarifies that Cldn7 plays an inhibitory role in CRC and reveals a possible molecular mechanism, which is conducive to further research on Cldn7 and cancer stem cells.

Generation and characterization of CD19-iCre mice as a tool for efficient and specific conditional gene targeting in B cells

The Cre/loxP system is a powerful tool for generating conditional gene knockout (KO) mice and elucidate gene function in vivo. CD19-Cre and Mb1-iCre transgenic mice are commonly used for generating B cell-specific KO mice and investigate the development, as well as the physiological and pathophysiological roles of B cells. However, the CD19-Cre line low efficiency and the Mb1-iCre line occasional ectopic recombination represent challenges for their use. Thus, we developed a CD19-codon-improved Cre (CD19-iCre) knock-in mouse with the T2A-iCre sequence inserted into the Cd19 locus, just before the stop codon. The CD19-iCre mice were compared with existing models, crossed with the Rosa26-EYFP reporter mice, and their recombination activity in B cells carrying different Cre alleles was assessed. CD19-iCre mice showed more effective Cre recombination in the early B cell developmental stages compared with the CD19-Cre mice. The efficiencies of the CD19-iCre and Mb1-iCre lines were similar; however, the B lineage-specific recombination was more stringent in the CD19-iCre line. Furthermore, the utility value of the CD19-iCre model was superior than that of the CD19-Cre mice regarding deletion efficiency in IL10-floxed mice. Thus, the CD19-iCre line is a valuable tool for highly efficient gene targeting specific to the B cell compartment.

Low-Dose Tamoxifen Induces Significant Bone Formation in Mice.

ABSTRACTUse of the selective estrogen receptor modulator Tamoxifen (TAM) is a mainstay to induce conditional expression of Cre recombinase in transgenic laboratory mice. To excise β‐cateninfl/fl in 28‐day‐old male and female Prrx1‐CreER/β‐cateninfl/fl mice (C57BL/6), we utilized TAM at 150 mg/kg; despite β‐catenin knockout in MSC, we found a significant increase in trabecular and cortical bone volume in all genders. Because TAM was similarly anabolic in KO and control mice, we investigated a dose effect on bone formation by treating wild‐type mice (WT C57BL/6, 4 weeks) with TAM (total dose 0, 20, 40, 200 mg/kg via four injections). TAM increased bone in a dose‐dependent manner analyzed by micro–computed tomography (μCT), which showed that, compared to control, 20 mg/kg TAM increased femoral bone volume fraction (bone volume/total volume [BV/TV]) (21.6% ± 1.5% to 33% ± 2.5%; 153%, p < 0.005). With TAM 40 mg/kg and 200 mg/kg, BV/TV increased to 48.1% ± 4.4% (223%, p < 0.0005) and 58% ± 3.8% (269%, p < 0.0001) respectively, compared to control. Osteoblast markers increased with 200 mg/kg TAM: Dlx5 (224%, p < 0.0001), Alp (166%, p < 0.0001), Bglap (223%, p < 0.0001), and Sp7 (228%, p < 0.0001). Osteoclasts per bone surface (Oc#/BS) nearly doubled at the lowest TAM dose (20 mg/kg), but decreased to <20% control with 200 mg/kg TAM. Our data establish that use of TAM at even very low doses to excise a floxed target in postnatal mice has profound effects on trabecular and cortical bone formation. As such, TAM treatment is a major confounder in the interpretation of bone phenotypes in conditional gene knockout mouse models. © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.

Oocyte-specific gene knockdown by intronic artificial microRNAs driven by Zp3 transcription in mice.

Conditional knockout technology is a powerful tool for investigating the spatiotemporal functions of target genes. However, generation of conditional knockout mice involves complicated breeding programs and considerable time. A recent study has shown that artificially designed microRNAs (amiRNAs), inserted into an intron of the constitutively expressed gene, induce knockdown of the targeted gene in mice, thus creating a simpler method to analyze the functions of target genes in oocytes. Here, to establish an oocyte-specific knockdown system, amiRNA sequences against enhanced green fluorescent protein (EGFP) were knocked into the intronic sites of the Zp3 gene. Knock-in mice were then bred with EGFP transgenic mice. Our results showed that Zp3-derived amiRNA successfully reduced EGFP fluorescence in the oocytes in a size-dependent manner. Importantly, knockdown of EGFP did not occur in somatic cells. Thus, we present our knockdown system as a tool for screening gene functions in mouse oocytes.

Cldn-7 deficiency promotes experimental colitis and associated carcinogenesis by regulating intestinal epithelial integrity

ABSTRACT Intestinal epithelial barrier protects intestine from infection and injury, while chronic inflammation is a trigger for tumorigenesis. As a member of tight junctions (TJs) family, Claudin-7 (Cldn-7) is dedicated to maintaining cell polarity and TJs barrier integrity, and closely related to the development of inflammation and tumors. However, potential roles of Cldn-7 in intestinal inflammation and colitis-associated colorectal cancer (CAC) have not been well characterized in vivo. Here, we analyzed the expression profile of Cldn-7 in inflammatory bowel disease (IBD) and CAC. Colitis and colitis-cancer transformation models were established based on inducible intestinal conditional Cldn-7 gene knockout mice (Cldn7fl/fl;villin-CreERT2), by intraperitoneal injection of azomethane (AOM) and dextran sodium sulfate (DSS) feeding. Cldn-7 knockout promoted susceptibility to colitis and CAC, aggravated clinical symptoms, severely damaged intestinal epithelium, increased mucosal inflammation accompanied dysregulated cell proliferation-apoptosis. Epithelial barrier integrity was destroyed, and intercellular permeability was increased. After AOM/DSS induction, tumor burden and volume were increased, characterized by enhanced proliferation and activation of Wnt/β-catenin signaling pathway. Mechanistically, Cldn-7 deficiency promoted colitis and subsequently malignant transformation by destroying TJs integrity and increasing inflammatory cascade. Overall, based on Cldn-7 knockout mouse model, we have first demonstrated the key roles of Cldn-7 in maintaining intestinal homeostasis and preventing IBD and consequent CAC. Abbreviations: AJs: adherens junctions; AOM: azomethane; Cldn-7: Claudin-7; CRC: colorectal cancer; CAC: colitis-associated colorectal cancer; CD: Crohn’s disease; DSS: dextran sodium sulfate; DAI: disease activity index; EMT: epithelial-mesenchymal transition; FITC: fluorescence isothiocyanate; HB: hemoglobin; IBD: inflammatory bowel disease; IECs: intestinal epithelial cells; ISCs: intestinal stem cells; PLT: platelet; RBC: red blood cell; ROS: reactive oxygen species; TAM: tamoxifen; TJs: tight junctions; TCF/LEF: T-cell factor/lymphoid enhancer factor; UC: ulcerative colitis; WBC: white blood cell.

Conditional knockout of the PDK-1 gene in osteoblasts affects osteoblast differentiation and bone formation.

Osteoblasts are the main functional cells of bone formation, and they are responsible for the synthesis, secretion, and mineralization of the bone matrix. Phosphatidylinositol-3-kinase/Akt is an important signaling pathway involved in the regulation of cell proliferation, death, and survival. Some studies have shown that 3-phosphoinositide-dependent protein kinase-1 (PDK-1) plays an important role in the phosphorylation of Akt. In the present study, an osteocalcin (OCN) promoter-driven Cre-LoxP system was established to specifically delete the PDK-1 gene in osteoblasts. It was found that the size and weight of PDK-1 conditional gene knockout (cKO) mice were significantly reduced. von Kossa staining and microcomputed tomography showed that the trabecular thickness, trabecular number, and bone volume were significantly decreased, whereas trabecular separation was increased, as compared with wide-type littermates, which were characterized by a decreased bone mass. A model of distal femoral defect was established, and it was found that cKO mice delayed bone defect repair. In osteoblasts derived from PDK-1 cKO mice, the alkaline phosphatase (ALP) secretion and ability of calcium mineralization were significantly decreased, and the expressions of osteoblast-related proteins, runt-related transcription factor 2, OCN, and ALP were also clearly decreased. Moreover, the phosphorylation level of Akt and downstream factor GSK3β and their response to insulin-like growth factor-1 (IGF-1) decreased clearly. Therefore, we believe that PDK-1 plays a very important role in osteoblast differentiation and bone formation by regulating the PDK-1/Akt/GSK3β signaling pathway.

Construction of fibronectin conditional gene knock-out mice and the effect of fibronectin gene knockout on hematopoietic, biochemical and immune parameters in mice.

Fibronectin (FN) is a multi-functional glycoprotein that primarily acts as a cell adhesion molecule and tethers cells to the extra cellular matrix. In order to clarify the effect of FN deficiency on hematopoiesis, biochemical and immune parameters in mice. We constructed a tamoxifen-induced conditional (cre-loxp system) fibronectin knock-out (FnKO) mouse model on a C57BL/6 background, and monitored their behavior, fertility, histological, hematopoietic, biochemical and immunological indices. We found that the Fn KO mice had reduced fertility, high platelet counts, smaller bone marrow megakaryocytes and looser attachment between the hepatocyte and vascular endothelial junctions compared to the wild type (WT) mice. In contrast, the behavior, hematological counts, serum biochemical indices and vital organ histology were similar in both Fn KO and WT mice. This model will greatly help in elucidating the role of FN in immune-related diseases in future.