Effects of Reduced Connexin43 Function on Mandibular Morphology and Osteogenesis in Mutant Mouse Models of Oculodentodigital Dysplasia
Mutations in the gene encoding the gap-junctional protein connexin43 (Cx43) is the cause of human disease oculodentodigital dysplasia (ODDD). mandible often affected by this disease, the clinical report describes both the overgrowth of the mandible and vice versa, retrognathia.
Observations were apparently opposed to underline our relative lack of understanding of how ODDD affect the morphology of the lower jaw. Using two mouse models that mimic the mutant phenotype ODDD (I130T / + and G60S / +), we sought to uncover how the altered function of Cx43 can affect the development of the lower jaw.
Specifically, jaw newborn mice were imaged using micro-CT, to allow statistical comparison of forms. network-level comparison of the key areas of the mandible is done by using histomorphology, and we quantified the mRNA expression of some of the cartilage and bone cell differentiation markers. Both G60S / + and I130T / + mice mutant has changed the morphology of the mandibular compared to their wildtype counterparts, and morphological effects are equally locally for both mutants.
In particular, the largest phenotypic differences in mutant mice focused in areas exposed to mechanical forces, such as alveolar bone, muscle attachment sites, and the articular surface. Histological analysis revealed differences intramembranous ossification of the jaw bone both mutant mice compared to their wildtype littermates. However, the organization of chondrocytes in cartilage secondary mandible is not affected in the mutant mice. Overall, our results indicate that morphological differences seen in the jaw G60S / + and I130T / + mouse as delayed ossification and suggested that mechanical forces can exacerbate the effects of ODDD bone.
Effects of Reduced Connexin43 Function on Mandibular Morphology and Osteogenesis in Mutant Mouse Models of Oculodentodigital Dysplasia
XIAP Protecting retinal ganglion cells in mutant ND4 Rat Model of Leber Hereditary Optic Neuropathy
Objective: Leber hereditary optic neuropathy (LHON) is a genetic form of vision loss that occurs mainly due to a mutation in the nicotinamide adenine dinucleotide dehydrogenase (ND) subunits that make up the complex I of the electron transport chain. LHON mutations resulted in the death of retinal ganglion cell apoptosis. We tested the hypothesis that gene therapy with X-linked inhibitor of apoptosis (XIAP) will prevent retinal ganglion cell apoptosis and reduce disease progression in a mouse model of LHON induced vector that carries a mutation ND4.
Methods: Adeno associated virus (AAV) encoding full-length hemagglutinin-tagged XIAP (AAV2.HA-XIAP) or green fluorescent protein (AAV2.GFP) injected into the vitreous of DBA / 1J mice. Two weeks later, LHON phenotype induced by AAV delivery of mutant ND4 (AAV2.mND4FLAG) into the vitreous. Retinal function was assessed by electroretinography pattern. Optic nerve harvested at 4 months, and XIAP therapeutic effect on the nerve fiber layer and optic nerve integrity was evaluated using immunohistochemistry, transmission electron microscopy and magnetic resonance imaging.
Description: Human kidney tissue lysate was prepared by homogenization using a proprietary technique. The tissue was frozen in liquid nitrogen immediately after excision and then stored at -70°C. The human kidney tissue total protein is provided in a buffer including HEPES (pH7.9), MgCl2, KCl, EDTA, Sucrose, Glycerol, Sodium deoxycholate, NP-40, and a cocktail of protease inhibitors. For quality control purposes, the kidney tissue pattern on SDS-PAGE gel is shown to be consistent for each lot by visualization with coomassie blue staining. The kidney tissue is then Western analyzed by either GAPDH or β-actin antibody, and the expression level is consistent with each lot.
Description: Multiple organ digestive system diseased tissue array with normal tissue as control, including TNM, clinical stage and pathology grade, 40 cases/40 cores
Genomic DNA - Human Diabetic Diseased Tissue: Bone Marrow, from a single donor
Description: Kidney cancer tissue array with adjacent normal kidney tissue, including TNM, clinical stage and pathology grade, 72 cases/72 cores, replacing BC07015a
Genomic DNA - Human Diabetic Diseased Tissue: Small Intestine: Jejunum, from a single donor
Description: Kidney cancer tissue array with matched adjacent normal kidney tissue, including TNM, clinical stage and pathology grade, 40 cases/90 cores, replacing KD901
All-in-One First-Strand cDNA Synthesis SuperMix for PCR
Description: The Matched Pair Paraffin Tissue (MPPT) slides are designed for identifying tumor-specific/metastasis genes or proteins. Slices from normal and malignant tissues are mounted on each MPPT slide which can then be treated as a single histological slide for H&E staining, immunohistochemistry, or in situ hybridization. This format allows a rapid analysis of protein expression and localization across normal and cancerous tissue.
Description: This cell lysate is prepared from rat kidney tissue using Boster's RIPA Lysis Buffer (AR0105) using a standard whole cell lysate protocol. The concentration was determined using the BCA assay process and then diluted using Dithiothreitol (DTT) and a reducing SDS sample loading buffer, heated for 5 minutes at 100˚C.
First.Strand cDNA PLUS Synthesis Kit (20 µl / reaction)
Description: Kidney tumor tissue lysate was prepared by homogenization in modified RIPA buffer (150 mM sodium chloride, 50 mM Tris-HCl, pH 7.4, 1 mM ethylenediaminetetraacetic acid, 1 mM phenylmethylsulfonyl fluoride, 1% Triton X-100, 1% sodium deoxycholic acid, 0.1% sodium dodecylsulfate, 5 μg/ml of aprotinin, 5 μg/ml of leupeptin. Tissue and cell debris was removed by centrifugation. Protein concentration was determined with Bio-Rad protein assay. The product was boiled for 5 min in 1 x SDS sample buffer (50 mM Tris-HCl pH 6.8, 12.5% glycerol, 1% sodium dodecylsulfate, 0.01% bromophenol blue) containing 5% β-mercaptoethanol.
Description: Kidney tumor tissue lysate was prepared by homogenization in modified RIPA buffer (150 mM sodium chloride, 50 mM Tris-HCl, pH 7.4, 1 mM ethylenediaminetetraacetic acid, 1 mM phenylmethylsulfonyl fluoride, 1% Triton X-100, 1% sodium deoxycholic acid, 0.1% sodium dodecylsulfate, 5 μg/ml of aprotinin, 5 μg/ml of leupeptin. Tissue and cell debris was removed by centrifugation. Protein concentration was determined with Bio-Rad protein assay. The product was boiled for 5 min in 1 x SDS sample buffer (50 mM Tris-HCl pH 6.8, 12.5% glycerol, 1% sodium dodecylsulfate, 0.01% bromophenol blue) containing 5% β-mercaptoethanol.
Description: Kidney tumor tissue lysate was prepared by homogenization in modified RIPA buffer (150 mM sodium chloride, 50 mM Tris-HCl, pH 7.4, 1 mM ethylenediaminetetraacetic acid, 1 mM phenylmethylsulfonyl fluoride, 1% Triton X-100, 1% sodium deoxycholic acid, 0.1% sodium dodecylsulfate, 5 μg/ml of aprotinin, 5 μg/ml of leupeptin. Tissue and cell debris was removed by centrifugation. Protein concentration was determined with Bio-Rad protein assay. The product was boiled for 5 min in 1 x SDS sample buffer (50 mM Tris-HCl pH 6.8, 12.5% glycerol, 1% sodium dodecylsulfate, 0.01% bromophenol blue) containing 5% β-mercaptoethanol.
Description: Kidney tumor tissue lysate was prepared by homogenization in modified RIPA buffer (150 mM sodium chloride, 50 mM Tris-HCl, pH 7.4, 1 mM ethylenediaminetetraacetic acid, 1 mM phenylmethylsulfonyl fluoride, 1% Triton X-100, 1% sodium deoxycholic acid, 0.1% sodium dodecylsulfate, 5 μg/ml of aprotinin, 5 μg/ml of leupeptin. Tissue and cell debris was removed by centrifugation. Protein concentration was determined with Bio-Rad protein assay. The product was boiled for 5 min in 1 x SDS sample buffer (50 mM Tris-HCl pH 6.8, 12.5% glycerol, 1% sodium dodecylsulfate, 0.01% bromophenol blue) containing 5% β-mercaptoethanol.
Description: Kidney tumor tissue lysate was prepared by homogenization in modified RIPA buffer (150 mM sodium chloride, 50 mM Tris-HCl, pH 7.4, 1 mM ethylenediaminetetraacetic acid, 1 mM phenylmethylsulfonyl fluoride, 1% Triton X-100, 1% sodium deoxycholic acid, 0.1% sodium dodecylsulfate, 5 μg/ml of aprotinin, 5 μg/ml of leupeptin. Tissue and cell debris was removed by centrifugation. Protein concentration was determined with Bio-Rad protein assay. The product was boiled for 5 min in 1 x SDS sample buffer (50 mM Tris-HCl pH 6.8, 12.5% glycerol, 1% sodium dodecylsulfate, 0.01% bromophenol blue) containing 5% β-mercaptoethanol.
Description: Kidney tumor tissue lysate was prepared by homogenization in modified RIPA buffer (150 mM sodium chloride, 50 mM Tris-HCl, pH 7.4, 1 mM ethylenediaminetetraacetic acid, 1 mM phenylmethylsulfonyl fluoride, 1% Triton X-100, 1% sodium deoxycholic acid, 0.1% sodium dodecylsulfate, 5 μg/ml of aprotinin, 5 μg/ml of leupeptin. Tissue and cell debris was removed by centrifugation. Protein concentration was determined with Bio-Rad protein assay. The product was boiled for 5 min in 1 x SDS sample buffer (50 mM Tris-HCl pH 6.8, 12.5% glycerol, 1% sodium dodecylsulfate, 0.01% bromophenol blue) containing 5% β-mercaptoethanol.
Description: Kidney tumor tissue lysate was prepared by homogenization in modified RIPA buffer (150 mM sodium chloride, 50 mM Tris-HCl, pH 7.4, 1 mM ethylenediaminetetraacetic acid, 1 mM phenylmethylsulfonyl fluoride, 1% Triton X-100, 1% sodium deoxycholic acid, 0.1% sodium dodecylsulfate, 5 μg/ml of aprotinin, 5 μg/ml of leupeptin. Tissue and cell debris was removed by centrifugation. Protein concentration was determined with Bio-Rad protein assay. The product was boiled for 5 min in 1 x SDS sample buffer (50 mM Tris-HCl pH 6.8, 12.5% glycerol, 1% sodium dodecylsulfate, 0.01% bromophenol blue) containing 5% β-mercaptoethanol.
Description: Kidney tumor tissue lysate was prepared by homogenization in modified RIPA buffer (150 mM sodium chloride, 50 mM Tris-HCl, pH 7.4, 1 mM ethylenediaminetetraacetic acid, 1 mM phenylmethylsulfonyl fluoride, 1% Triton X-100, 1% sodium deoxycholic acid, 0.1% sodium dodecylsulfate, 5 μg/ml of aprotinin, 5 μg/ml of leupeptin. Tissue and cell debris was removed by centrifugation. Protein concentration was determined with Bio-Rad protein assay. The product was boiled for 5 min in 1 x SDS sample buffer (50 mM Tris-HCl pH 6.8, 12.5% glycerol, 1% sodium dodecylsulfate, 0.01% bromophenol blue) containing 5% β-mercaptoethanol.
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Results: During the development of LHON disease, retinal ganglion cell axons lost. Body core apoptotic cells seen in astrocytes or oligodendrocytes in the optic nerve, and there is thinning of the optic nerve and retinal nerve fiber layer. At 4 months after the onset of illness, XIAP gene therapy protects the nerve fiber layer and optic nerve architecture by maintaining healthy axons. XIAP also decreased nuclear fragmentation in the population of astrocytes or oligodendrocytes and reduce infiltration of glial cells.
