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)

Muscular dysgenesis (mdg) is a recessive lethal mutation in the mouse which drastically affects skeletal muscle development during embryonic life. Physiologically, the disease is characterized by a complete paralysis resulting from a lack of excitation-contraction coupling. Existing electrophysiological, biochemical, and genetic evidence shows that mdg/mdg mice express a basic alteration of L-type voltage-sensitive Ca2+ channels in skeletal muscle. Studies on mdg/mdg myotubes in primary culture have shown that +/+ fibroblasts or +/+ Schwann cells may fuse with them and correct their functional deficiency by genetic complementation. As the spontaneous formation of heterocaryons is thought to be an exclusive property of myoblasts, we asked whether fibroblasts may have changed their properties before fusion occurred. We used primary cells issued from sciatic nerves dissected from newborn transgenic mice carrying the pHuDes1-nls-LacZ transgene (Des-LacZ cells) as non-muscle cells. These cells were mainly fibroblasts (80%) positive for Thy1.1 and Schwann cells positive for S100. The cultures were negative for myogenic markers (desmin, troponin T), did not form myotubes long-term, and did not display significant activation of the muscle reporter gene (pHuDes1-nls-LacZ). After a few days in coculture with dysgenic or normal myotubes, the muscle reporter gene (beta-galactosidase) was detected both within dysgenic myotubes, correlating with the restoration of normal contractile activity, and normal myotubes. As well as confirming that fusion takes place, this shows that Des-LacZ cells nuclei incorporated into recipient myotubes express their own myogenic genes. Moreover, individual mononucleated Des-LacZ cells expressing beta-galactosidase were observed, indicating that myogenic genes were being expressed before fusion. This suggests a mechanism of myotube driven myogenic recruitment of cells during the in vitro myogenesis. Analysis of the distribution of the induced Des-LacZ cells (positive for beta-galactosidase) in compartmentalized muscle cocultures showed that in the presence of dysgenic myotubes, these cells were equally distributed in both myotube free and enriched areas, whereas in the presence of normal myotubes, the positive cells remained in close vicinity of the myotubes. This difference could be explained by the fact that the dysgenic phenotype might include release of the induction process from its normal controls. Our results are consistent with the idea of a transcellular mechanism triggering myogenic differentiation in non-muscle cells, and that myotubes themselves are able to drive myogenic recruitment of cells during the in vitro myogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Myotube driven myogenic recruitment of cells during in vitro myogenesis. 773 31

The transplantation of cultured myoblasts into mature skeletal muscle is the basis for a new therapeutic approach to muscle and non-muscle diseases: myoblast-mediated gene therapy. The success of myoblast transplantation for correction of intrinsic muscle defects depends on the fusion of implanted cells with host myofibers. Previous studies in mice have been problematic because they have involved transplantation of established myogenic cell lines or primary muscle cultures. Both of these cell populations have disadvantages: myogenic cell lines are tumorigenic, and primary cultures contain a substantial percentage of non-myogenic cells which will not fuse to host fibers. Furthermore, for both cell populations, immune suppression of the host has been necessary for long-term retention of transplanted cells. To overcome these difficulties, we developed novel culture conditions that permit the purification of mouse myoblasts from primary cultures. Both enriched and clonal populations of primary myoblasts were characterized in assays of cell proliferation and differentiation. Primary myoblasts were dependent on added bFGF for growth and retained the ability to differentiate even after 30 population doublings. The fate of the pure myoblast populations after transplantation was monitored by labeling the cells with the marker enzyme beta-galactosidase (beta-gal) using retroviral mediated gene transfer. Within five days of transplantation into muscle of mature mice, primary myoblasts had fused with host muscle cells to form hybrid myofibers. To examine the immunobiology of primary myoblasts, we compared transplanted cells in syngeneic and allogeneic hosts. Even without immune suppression, the hybrid fibers persisted with continued beta-gal expression up to six months after myoblast transplantation in syngeneic hosts. In allogeneic hosts, the implanted cells were completely eliminated within three weeks. To assess tumorigenicity, primary myoblasts and myoblasts from the C2 myogenic cell line were transplanted into immunodeficient mice. Only C2 myoblasts formed tumors. The ease of isolation, growth, and transfection of primary mouse myoblasts under the conditions described here expand the opportunities to study muscle cell growth and differentiation using myoblasts from normal as well as mutant strains of mice. The properties of these cells after transplantation--the stability of resulting hybrid myofibers without immune suppression, the persistence of transgene expression, and the lack of tumorigenicity--suggest that studies of cell-mediated gene therapy using primary myoblasts can now be broadly applied to mouse models of human muscle and non-muscle diseases.
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PMID:Primary mouse myoblast purification, characterization, and transplantation for cell-mediated gene therapy. 820 57

We have constructed two sets of Escherichia coli lacZ-based vectors for use in studies of general mitotic recombination, both in somatic mammalian cells grown in culture and in transgenic animals. The vectors use two mutant copies of the E. coli lacZ gene as their recombination substrates and contain a neo gene for selection of stable transformants. In one vector, pLrec, an SV40 promoter drives lacZ, while the other vector, pArec, utilizes a human non-muscle beta-actin promoter for lacZ expression. Gene conversions, unequal sister chromatid exchanges and reciprocal exchanges between the two lacZ genes result in expression of beta-galactosidase, which can be detected in situ by histochemical staining. These vectors yield rates and frequencies of mitotic intrachromosomal recombination in human and rodent cell lines which are similar to rates reported using conventional recombination vectors. Molecular analysis of recombinational events involving the lacZ-based vectors can be carried out on genomic DNA isolated from clonally expanded populations and individual LacZ+ cells and cell clusters can be analyzed using PCR amplification. These reporter gene-based vectors may facilitate the study of recombination in cells with limited proliferative capacities, allow for analysis of both products of an unequal sister chromatid exchange, and permit in situ detection of recombination in the tissues of transgenic animals.
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PMID:Novel lacZ-based recombination vectors for mammalian cells. 829 44

The dystrophin gene, which is defective in Duchenne muscular dystrophy (DMD), also encodes a number of smaller products controlled by internal promoters. Dp71, which consists of the two C-terminal domains of dystrophin, is the most abundant product of the gene in non-muscle tissues and is the major product in adult brain. To study the possible function of Dp71 and its expression during development, we specifically inactivated the expression of Dp71 by replacing its first and unique exon and a part of the concomitant intron with a beta-galactosidase reporter gene. X-Gal staining of Dp71-null mouse embryos and tissues revealed a very stage- and cell type-specific activity of the Dp71 promoter during development and during differentiation of various tissues, including the nervous system, eyes, limb buds, lungs, blood vessels, vibrissae and hair follicles. High activity of the Dp71 promoter often seemed to be associated with morphogenic events and terminal differentiation. In some tissues the activity greatly increased towards birth.
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PMID:Targeted inactivation of Dp71, the major non-muscle product of the DMD gene: differential activity of the Dp71 promoter during development. 988 26

To investigate the function of the major non-muscle dystrophin isoform, Dp71, we substituted a beta-galactosidase (betagal) reporter gene for Dp71 by homologous recombination in embryonic stem cells. Staining for betagal activity in chimeric mice revealed Dp71 promoter activity in glial cells in the CNS, in neurons of the inner nuclear and inner plexiform layers of the retina, and in the kidney tubules and collecting ducts. Our observations demonstrate that Dp71 is widely expressed in the adult CNS (retina, cerebellum, cerebral cortex, ependyma, and choroid) as well as the adult kidney epithelium and suggest a broad function for Dp71 in differentiated tissues.
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PMID:Expression of the 71 kDa dystrophin isoform (Dp71) evaluated by gene targeting. 1035 May 71