Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.23 (beta-galactosidase)
14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Defective avian leukosis virus (ALV)-based vectors expressing the neo and LacZ genes were constructed under the control of cis-acting elements originated from 4 avian retroviruses: avian erythroblastosis virus (AEV), Rous associated viruses 1 (RAV-1) and 2 (RAV-2), and the Schmidt Ruppin strain of Rous sarcoma virus subgroup D (SR-RSV-D). We used these vectors to study the long-term stability of beta-galactosidase expression (encoded by the LacZ gene) in a permanent cell line from quail fibroblasts (QT6). Infection of the immortalized QT6 cell line with these vectors resulted in unstable beta-galactosidase expression. We determined whether this instability of provirus expression was correlated with: (1) presence of G418 selection; (2) deletion in the proviral genome; (3) hypermethylation of the proviral genome; (4) position of the neo and LacZ genes in the proviral genome; and (5) the transcriptional activity of the long terminal repeat (LTR) elements of proviral vectors. We observed that G418 selection pressure applied to infected QT6 cells lead to a more stable LacZ gene expression. Moreover, our results suggest a correlation between the stability of proviral gene expression and the level of gene expression driven by the LTR elements and depending on the strain origin of these.
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PMID:Influence of expression and cis-acting sequences from avian leukosis viruses (ALVs) on stability of (ALV)-based retrovirus vectors. 767 Oct

A catechol 2,3-dioxygenase (C23O) gene of Pseudomonas aeruginosa was expressed under the Simian virus 40 or Rous sarcoma virus promoter in mammalian cells; it was found that the gene could be used as a reporter for the study of gene expression. The C23O gene was a more sensitive reporter than the generally used beta-galactosidase gene.
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PMID:A catechol 2,3-dioxygenase gene as a reporter. 776 30

The efficacy of adenovirus (ADV)-mediated gene therapy to treat brain tumors was tested in a syngeneic glioma model. Tumor cells were transduced in situ with a replication-defective ADV carrying the herpes simplex virus thymidine kinase (HSV-tk) gene controlled by the Rous sarcoma virus promoter. Expression of the HSV-tk gene enables the transduced cell to convert the drug ganciclovir to a form that is cytotoxic to dividing cells. Tumors were generated in Fischer 344 rats by stereotaxic implantation of 9L gliosarcoma cells into the caudate nucleus. Eight days later, the tumors were injected either with the ADV carrying the HSV-tk (ADV-tk) gene or a control ADV vector containing the beta-galactosidase (ADV-beta gal) gene and the rats were treated with either ganciclovir or saline. Tumor size was measured 20 days after implantation of 9L cells or at death. Rats treated with ADV-beta gal and ganciclovir or with ADV-tk and saline had large tumors. No tumors were detected in animals treated with ADV-tk and with ganciclovir at doses > or = 80 mg/kg. An infiltrate of macrophages and lymphocytes at the injection site in animals treated with ADV-tk and ganciclovir indicated an active local immune reaction. In survival studies, all animals treated with ADV-tk and ganciclovir have remained alive longer than 80 and up to 120 days after tumor induction whereas all untreated animals died by 22 days. These results demonstrate that ADV-mediated transfer of HSV-tk to glioma cells in vivo confers sensitivity to ganciclovir, and represents a potential method of treatment of brain tumors.
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PMID:Adenovirus-mediated gene therapy of experimental gliomas. 788 26

In embryogenesis, avian primordial germ cells (PGCs) circulate temporarily in the blood vessels at stages 10-15 (Hamburger and Hamilton, 1951), before reaching the gonads. In an attempt to transfer cloned genes into PGCs, liposome consisting of reporter plasmid DNA and N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammoniummethylsulf ate was injected into the marginal veins of embryos at stages 11-15. As reporter plasmids, pRSVZ and pAcZ harboring the Escherichia coli lacZ gene driven, respectively, by the Rous sarcoma virus (RSV) promoter and the chicken beta-actin gene promoter were used. First, 55 embryos were injected with liposome containing pRSVZ and stained for the bacterial beta-galactosidase activity 24 hr after injection. In all the embryos, cells positive for beta-galactosidase activity were observed among the blood cells, endothelial cells, and endocardium cells of the heart, suggesting that transfection took place within the circulatory system. Then, embryos were injected with liposome containing pRSVZ or pAcZ, and stained 2 or 3 d after injection. PGCs positive for beta-galactosidase activity were observed in the gonads in four out of 44 embryos injected with pRSVZ, and 29 out of 71 embryos injected with pAcZ, indicating that the plasmid DNA was transferred into PGCs developing normally. The average number of positive PGCs per embryo was 0.2 and 2.1, respectively, when pRSVZ and pAcZ were introduced. The difference in the number of positive PGCs detected after introduction of the two plasmids suggests that the actin promoter has a higher level of transcriptional activity in PGCs than does the RSV promoter.
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PMID:Liposome-mediated DNA transfer into chicken primordial germ cells in vivo. 791 78

The best way to overcome immunorejection in heterologous myoblast transfer (HMT) is by the use of immunodeficient and/or highly immunosuppressed mice as hosts. The same may be attained by autologous myoblast transfer (AMT). In this paper, we describe myoblast transfer in mdx and normal mice where the donor myogenic cells originated from highly inbred litter mates that are considered to be isogenic and thus the procedure is analogous to AMT. The myoblasts were marked in vitro with Rous Sarcoma Virus (RSV)-luciferase (Lux) or RSV-beta-galactosidase (LacZ) reporter genes through transduction mediated by an autonomously replication-defective recombinant human adenovirus. This permitted us to follow their fate after transplantation. mdx and normal mice were irradiated with 20 Gray gamma rays; necrosis and regeneration were induced by intramuscular notexin prior to myoblast injection. In both mdx and normal mice, the expression of luciferase rapidly declined after the injection implying that a large portion of the injected myoblasts were lost by 48 hr, due to undetermined cause(s). The surviving, injected myoblasts well-mosaicized large groups of host fibers but only in the immediate vicinity of the injection. Substantial expression of the reporter gene continued up to 1 month post-transplantation in normal mice, but there was a gradual decline and eventual disappearance of the reporter gene expression in mdx mice. This latter phenomenon was due to the ongoing intense necrosis of muscle fibers in mdx. There was no evidence of immunorejection. These experiments indicate that even in the absence of immunorejection, myoblast transfer suffers from important negative features: major loss of myoblasts within 48 hr after the injection and lack of significant spread of the injected cells from the injection site in the host muscle. These factors, plus the limited proliferative and fusion capacity of Duchenne muscular dystrophy (DMD) myoblasts, make them less than an ideal vector for the dystrophin cDNA for dystrophin gene replacement therapy in DMD.
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PMID:Gene transfer into skeletal muscles by isogenic myoblasts. 794 44

Gene transfer vectors based on adeno-associated virus (AAV) are emerging as highly promising for use in human gene therapy by virtue of their characteristics of wide host range, high transduction efficiencies, and lack of cytopathogenicity. To better define the biology of AAV-mediated gene transfer, we tested the ability of an AAV vector to efficiently introduce transgenes into nonproliferating cell populations. Cells were induced into a nonproliferative state by treatment with the DNA synthesis inhibitors fluorodeoxyuridine and aphidicolin or by contact inhibition induced by confluence and serum starvation. Cells in logarithmic growth or DNA synthesis arrest were transduced with vCWR:beta gal, an AAV-based vector encoding beta-galactosidase under Rous sarcoma virus long terminal repeat promoter control. Under each condition tested, vCWR:beta Gal expression in nondividing cells was at least equivalent to that in actively proliferating cells, suggesting that mechanisms for virus attachment, nuclear transport, virion uncoating, and perhaps some limited second-strand synthesis of AAV vectors were present in nondividing cells. Southern hybridization analysis of vector sequences from cells transduced while in DNA synthetic arrest and expanded after release of the block confirmed ultimate integration of the vector genome into cellular chromosomal DNA. These findings may provide the basis for the use of AAV-based vectors for gene transfer into quiescent cell populations such as totipotent hematopoietic stem cells.
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PMID:Efficient gene transfer into nondividing cells by adeno-associated virus-based vectors. 805 46

To express high levels of proteins encoded by transfected DNA constructs in a variety of cultured cells, including neuronal cells, the activities of nine different promoters were evaluated using Escherichia coli beta-galactosidase (beta-gal) (LacZ) as a reporter gene. These nine promoters were categorized into three distinct groups (high, intermediate, and low expresser), in terms of the levels of beta-gal expression. An expression vector containing the cytomegalovirus enhancer and the chick beta-actin promoter (high expresser) showed the highest levels of expression, followed by vectors containing the cytomegalovirus promoter/enhancer and the SV40 promoter/enhancer (intermediate expresser). The rest of the promoters (thymidine kinase, adenovirus, murine proliferative sarcoma virus, nerve growth factor receptor, Rous sarcoma and mouse mammary tumor virus, and beta-amyloid precursor protein) expressed low levels of beta-gal. These results were consistent for eight different cell types. A particularly attractive model is the stem cell, P19; cultures differentiating into progeny consisting predominantly of cholinergic neurons could be readily transfected with expression vectors using liposomes and expressed beta-gal without significant morphologic changes of the differentiated neurons. The systems should be useful for the study of promoters and various expressed proteins, including those involved in axonal transport.
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PMID:Activity assays of nine heterogeneous promoters in neural and other cultured cells. 806 55

An avian hepatoma cell line has been reported to be suitable for the cultivation of avian laryngotracheitis virus (ILTV) (Scholz et al. (1993) J. Virol. Methods, 273-286; Guo et al. (1993) Am. J. Vet. Res., in press). To provide information for the establishment of avian expression systems and for the construction of avian recombinant viruses, five expression plasmids were constructed to test two avian viral and two mammalian viral promotors for their suitability and strength for gene expression in this cell line. Chicken hepatoma cells were transfected with plasmids carrying the bacterial beta-galactosidase (beta-gal) gene as a reporter gene. The beta-gal gene of three plasmid constructs expressed in both E. coli and avian hepatoma cells, while the beta-gal gene of two other constructs expressed only in avian hepatoma cells. The beta-gal gene expressed independently of any viral infection when under the control of the early Rous sarcoma virus (RSV) promoter or the immediate-early cytomegalovirus (CMV) promoter. However, expression of beta-gal gene under the control of the SV40 early promoter/enhancer and the ILTV TK promoter was greatly potentiated when the transfected cells were co-infected with ILTV. This finding provides a system for the enhancement of gene expression in avian cells, especially when ILTV is used as vector.
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PMID:Transactivation of the early SV40 promoter by avian infectious laryngotracheitis virus in avian hepatoma cells. 810 2

We have optimized a lipospermine-based transfection method for introducing genes into intact vertebrate embryos in vivo. The method employs small amounts of the cationic lipid Transfectam (DOGS), in a concentrated (40 mM) ethanolic solution, to compact and to transfer exogenous genes into chick embryos during the early stages of development (< 36 h of incubation). Plasmid vectors containing the reporter gene luciferase were used to follow the time course of expression. Luciferase activity was detected as early as 12 h post-transfection and was highest at this time. Enzyme activity then decreased over the next two days and was usually undetectable by 72-h post-transfection. To follow the spatial expression of the exogenous genes, a Rous sarcoma virus (RSV)-beta-galactosidase vector was used. When the transfection complex was applied externally around the developing embryo, the main site of expression was the cardiac tissue. Expression could be targeted to the nervous system by micro-injecting the DNA/DOGS (DNA/dioctadecylamidoglycylspermine) complex into the developing brain. The results show that reporter genes can be efficiently expressed in both the developing central nervous system and heart. This raises the possibility that lipospermines can be used to transfer functional genes into embryos during defined periods of development and also to deliver genes in other species and in other in vivo contexts.
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PMID:Temporal and spatial expression of lipospermine-compacted genes transferred into chick embryos in vivo. 818 24

A system for targeting foreign DNA to epithelial cells in vitro has been developed by exploiting receptor-mediated endocytosis. The polymeric immunoglobulin receptor transports dimeric immunoglobulin A and immunoglobulin M through epithelial cells, including those of the respiratory tract, by binding the immunoglobulins at the basolateral surface and transporting them across the cell. Fab fragments of antibodies directed against the extracellular portion of the receptor, secretory component, are similarly transported. Anti-human secretory component Fab fragments were covalently linked to a polycation, and complexed to various expression plasmids. When bound to an expression plasmid containing the Escherichia coli lacZ gene ligated to the Rous sarcoma virus promoter, the complexes transfected HT29.74 human colon carcinoma cells induced to express polymeric immunoglobulin receptor, but not those lacking the receptor. Primary cultures of human tracheal epithelial cells grown on collagen gels, which induce the expression of polymeric immunoglobulin receptor, were also transfected with the complexes. From 5 to 66% of the respiratory epithelial cells had beta-galactosidase activity after treatment, comparable to the percentage of cultured human tracheal epithelial cells that express polymeric immunoglobulin receptor (8-35%). The addition of excess human secretory component (Fab ligand) to the culture medium at the time of transfection blocked the delivery of DNA. The expression plasmid, either alone, complexed to the polycation, or complexed to a carrier based on an irrelevant Fab fragment, was not effective in transfecting either cell type. This DNA carrier system introduces DNA specifically into epithelial cells that contain pIgR in vitro.
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PMID:Gene transfer into respiratory epithelial cells by targeting the polymeric immunoglobulin receptor. 822 56


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