Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.2.1.23 (beta-galactosidase)
 14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In man, deficiency of ADA activity is associated with an autosomal recessive form of severe combined immunodeficiency (SCID), a disease with profound defects both cellular and humoral immunity. Current treatments of ADA deficient patients include bone marrow transplantation, enzyme replacement and somatic gene therapy. The mechanism of the selective immune cell pathogenesis in ADA-SCIDS is, however, still poorly understood. Thus, the generation of an ADA deficient mouse model will be of considerable benefit to understand better the pathophysiology of the disorder and to improve the gene therapy treatments. We have disrupted the adenosine deaminase (ADA) gene in embryonic stem cells using a new efficient promoter trap gene-targeting approach. To this end, a dicistronic targeting construct containing a promoterless IRES beta geo cassette was used. This cassette allows, via the internal ribosomal entry site (IRES), the direct cap-independent translation of the beta geo reporter gene which encodes a protein with both beta-galactosidase and neomycin activities. After indentification of targeted clones by Southern blot, successful inactivation of the ADA gene was first confirmed by producing, from our heterozygote clones, an homozygote cell line. This line shows no ADA activity as judged by zymogram analysis. Second, we have been able to detect in the targeted clones, a specific beta galactosidase activity using a sensitive fluorogenic assay. The targeted ES cell clones are currently being injected into blastocysts to create an ADA deficient mouse model.
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PMID:Disruption of the adenosine deaminase (ADA) gene using a dicistronic promoterless construct: production of an ADA-deficient homozygote ES cell line. 765 14

DNA-based immunization of mice by intramuscular injection of antigen-encoding plasmid DNA results in immune responses which may be sustained for extended periods of time without an antigen boost. For example, we have previously shown that a strong humoral response against hepatitis B virus surface antigen (HBsAg) will persist for up to 74 weeks following a single intramuscular administration of DNA. It has been proposed that the longevity of the response is due to sustained expression of antigen in transfected muscle cells. However, here we show by immunohistochemistry and electron microscopy that HBsAg-expressing muscle fibers are destroyed around 10 days after injection of DNA in mice. We have also evaluated destruction of the transfected muscle fibers indirectly, by measurement of luciferase activity in muscles at different times after injection of a luciferase reporter gene construct, alone or in combination with HBsAg-expressing DNA. Control muscles injected with luciferase-expressing DNA alone maintain expression of high levels of luciferase for at least 60 days. In contrast, muscles co-injected with DNAs expressing luciferase and a secreted form of HBsAg show high levels of luciferase activity at 5 days but > 99% of this is lost by 20 days. Similar results are obtained with co-expression of luciferase and beta-galactosidase, a non-secreted antigen. Loss of luciferase expression does not occur in muscles of mice with severe combined immunodeficiency, indicating that the myofiber destruction is immunologically mediated.
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PMID:Immune-mediated destruction of transfected muscle fibers after direct gene transfer with antigen-expressing plasmid DNA. 913 31

Ex vivo gene therapy of primary myopathies, based on autologous transplantation of genetically modified myogenic cells, is seriously limited by the number of primary myogenic cells that can be isolated, expanded, transduced, and reimplanted into the patient's muscles. We explored the possibility of using the MyoD gene to induce myogenic conversion of nonmuscle, primary cells in a quantitatively relevant fashion. Primary human and murine fibroblasts from skin, muscle, or bone marrow were infected by an E1-deleted adenoviral vector carrying a retroviral long terminal repeat-promoted MyoD cDNA. Expression of MyoD caused irreversible withdrawal from the cell cycle and myogenic differentiation in the majority (from 60 to 90%) of cultured fibroblasts, as defined by activation of muscle-specific genes, fusion into contractile myotubes, and appearance of ultrastructurally normal sarcomagenesis in culture. 24 h after adenoviral exposure, MyoD-converted cultures were injected into regenerating muscle of immunodeficient (severe combined immunodeficiency/beige) mice, where they gave rise to beta-galactosidase positive, centrally nucleated fibers expressing human myosin heavy chains. Fibers originating from converted fibroblasts were indistinguishable from those obtained by injection of control cultures of lacZ-transduced satellite cells. MyoD-converted murine fibroblasts participated to muscle regeneration also in immunocompetent, syngeneic mice. Although antibodies from these mice bound to adenoviral infected cells in vitro, no inflammatory infiltrate was present in the graft site throughout the 3-wk study period. These data support the feasibility of an alternative approach to gene therapy of primary myopathies, based on implantation of large numbers of genetically modified primary fibroblasts massively converted to myogenesis by adenoviral delivery of MyoD ex vivo.
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PMID:High efficiency myogenic conversion of human fibroblasts by adenoviral vector-mediated MyoD gene transfer. An alternative strategy for ex vivo gene therapy of primary myopathies. 959 68

A 60-Mb murine chromosome consisting of murine pericentric satellite DNA and two bands of integrated marker and reporter genes has been generated de novo in a rodent/human hybrid cell line (mM2C1). This prototype mammalian artificial chromosome platform carries a normal centromere, and the expression of its beta-galactosidase reporter gene has remained stable under selection for over 25 months. The novel chromosome was transferred by a modified microcell fusion method to mouse [L-M(TK-)], bovine (P46) and human (EJ30) cell lines. In all cases, the chromosome remained structurally and functionally intact under selection for periods exceeding 3 months from the time of transfer into the new host. In addition, the chromosome was retained in three first-generation tumours when L-M(TK-) cells containing the chromosome were xenografted in severe combined immunodeficiency mice. These data support that a murine satellite DNA-based artificial chromosome can be used as a functional mammalian artificial chromosome and can be maintained in vivo and in cells of heterologous species in vitro.
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PMID:Stability of a functional murine satellite DNA-based artificial chromosome across mammalian species. 1021 27

Generation of a vascular network is a hallmark of solid tumor growth, and attempts to switch off the tumor angiogenic phenotype are promising. However, this angiogenic potential might also be exploited to obtain incorporation into tumor vessels of genetically modified third-party cells, which could behave as targets of immunologic or pharmacologic attack. With this in mind, we addressed the efficiency and selectivity of third-party cell recruitment into experimental tumors generated in severe combined immunodeficiency mice. The animals were inoculated intraperitoneally with human ovarian carcinoma cell lines and with beta-galactosidase (beta-gal)-transduced human umbilical vein endothelial cell (HUVEC) or human fibroblasts. Transgenic HUVEC were scattered in tumors, but not in normal mouse tissues; immunohistochemical analysis revealed their selective homing to tumor vascular structures, over 50% of which contained beta-gal(+) cells. Injection of beta-gal-transduced human fibroblasts was also associated with transgenic cell incorporation into tumor masses; however, beta-gal(+) fibroblasts did not home to tumor blood vessels and were only localized within the tumor stroma. These findings show that the recruitment of primary third-party cells into the different compartments of experimentally induced tumors is an efficient and selective phenomenon and indicate possible alternative ways of confronting the tumor angiogenic potential in cancer therapy.
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PMID:Recruitment of human umbilical vein endothelial cells and human primary fibroblasts into experimental tumors growing in SCID mice. 1279 79

CRACM1 (also called Orai1) constitutes the pore subunit of store-operated calcium release-activated calcium channels. A point mutation in the gene encoding CRACM1 is associated with severe combined immunodeficiency disease in humans. Here we generated CRACM1-deficient mice in which beta-galactosidase activity 'reported' CRACM1 expression. CRACM1-deficient mice were smaller in size. Mast cells derived from CRACM1-deficient mice showed grossly defective degranulation and cytokine secretion, and the allergic reactions elicited in vivo were inhibited in CRACM1-deficient mice. We detected robust CRACM1 expression in skeletal muscles and some regions of the brain, heart and kidney but not in the lymphoid regions of thymus and spleen. In contrast, we found CRACM2 expression to be much higher in mouse T cells. In agreement with those findings, the store-operated calcium influx and development and proliferation of CRACM1-deficient T cells was unaffected. Thus, CRACM1 is crucial in mouse mast cell effector function, but mouse T cell calcium release-activated calcium channels are functional in the absence of CRACM1.
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PMID:Defective mast cell effector functions in mice lacking the CRACM1 pore subunit of store-operated calcium release-activated calcium channels. 1805 70