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:1.14.14.3 (luciferase)
38,195 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We determined the time course of gene expression following DNA/Lipofectin transfection of normal or previously injured arterial segments using direct intraluminal infusion following surgical exposure. Constructs possessing the firefly luciferase cDNA regulated by Simian virus 40, Rous sarcoma virus, or alpha-actin promoter were incubated together with Lipofectin for 30 minutes. Arterial segments were assayed for luciferase activity following harvest at 2-21 days. Without prior injury, luciferase activity was only 2.5-fold greater than background two days following gene transfer. Arterial injury three days before gene transfer resulted in luciferase activity 12.5-fold over background levels. This observation has clinical implications with regard to gene therapy following angioplasty, a procedure that is associated with endothelial cell denudation and smooth muscle cell proliferation. Maintenance of gene expression for several days could ameliorate the early smooth muscle migration and proliferation following arterial injury.
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PMID:Prior arterial injury enhances luciferase expression following in vivo gene transfer. 838 Jun 95

The present study demonstrates that the human adenovirus (Ad) can augment transfer and expression of a gene within plasmid DNA unmodified by nonspecific linkers or by linker-ligand complexes. Following the transfection of COS-7 cells with pRSVL, a luciferase expression plasmid vector directed by the Rous sarcoma virus-long terminal repeat promoter, luciferase activity in the target cells was 10(3)- to 10(4)-fold higher when the cells were also infected with Ad-CFTR, a replication-deficient recombinant Ad containing human cystic fibrosis transmembrane conductance regulator (CFTR) cDNA. The enhancement of luciferase gene expression in COS-7 cells was also observed with Ad-dl312 (a replication-deficient E1a deletion mutant Ad with no exogenous gene) and wild type Ad5. The efficiency of cell transfection with pRSVL in the presence of an Ad was achieved in a dose-dependent fashion with progressively higher luciferase activity in cells infected by increasing amounts of Ad-CFTR, Ad-dl312, or Ad5. The augmentation by Ad-CFTR of the transfer and expression of the luciferase gene in cells was similar to that of another transfection reagent, cationic liposomes. Further, when Ad-CFTR and liposomes were used in combination, 4- to 100-fold more efficient expression of the luciferase gene was achieved than with Ad-CFTR or liposomes alone. When COS-7, HeLa, and CV-1 cells were evaluated in parallel in the presence or absence of liposomes, Ad-mediated enhancement of luciferase activity was observed in all cell lines. Thus, exposure of target cells to replication-deficient or competent human Ad will markedly augment transfer and expression of the genes within plasmid DNA in mammalian cells in vitro without modifying the plasmid with linkers or linker-ligand complexes, a strategy that should be useful for in vitro and in vivo gene transfer applications.
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PMID:Adenovirus-mediated augmentation of cell transfection with unmodified plasmid vectors. 842 4

We have investigated the regulated expression of genes injected into the heart of large mammals in situ. Reporter constructs using the chloramphenicol acetyltransferase gene under the control of muscle-specific beta-myosin heavy chain (beta-MHC) or promiscuous (mouse sarcoma virus) promoters were injected into the canine myocardium. There was a linear dose-response relation between the level of gene expression and the quantity of plasmid DNA injected between 10 and 200 micrograms per injection site. The level of reporter gene expression did not correlate with the amount of injury imposed on the cardiac tissue. There was no regional variation in expression of injected reporter genes throughout the left ventricular wall. By use of both the mouse sarcoma virus and a muscle-specific beta-MHC promoter, reporter gene expression was one to two orders of magnitude greater in the heart than in skeletal muscle. Expression in the left ventricle was threefold higher than in the right ventricle. Chloramphenicol acetyltransferase activity was detected at 3, 7, 14, and 21 days after injection, with maximal expression at 7 days after injection. Statistical analysis of coinjection experiments revealed that coinjection of a second gene construct (Rous sarcoma virus-luciferase) is useful in the control of transfection efficiency in vivo. Furthermore, using reporter constructs containing serial deletions of the 5' flanking region of the beta-MHC gene, we performed a series of experiments that demonstrate the utility of this model in mapping promoter regions and identifying important regulatory gene sequences in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Gene injection into canine myocardium as a useful model for studying gene expression in the heart of large mammals. 843 91

Striated muscle is the only tissue found to be capable of taking up and expressing reporter genes that are transferred in the form of plasmid DNA. Thus, direct gene transfer is a potential method of gene therapy for the primary inherited myopathies. However, results to date have had insufficient and too variable expression to consider using direct gene transfer in human trials. We have determined that much of the variability of expression is due to nonuniform distribution of substances injected into skeletal muscle in vivo, and have developed a model to ameliorate this. Preinjection of muscles with a relatively large volume of hypertonic sucrose improves the distribution of injected substances and results in significantly less variable expression of reporter genes for luciferase or beta-galactosidase; the coefficient of variation for mean luciferase activity was reduced from about 120% to 25%. Expression is not directly proportional to dose, but is more so if the muscles are preinjected with sucrose than not. Expression is higher and less variable if DNA is injected in a larger than a smaller volume. The choice of promoter appears to be particularly important. Luciferase reporter gene expression from the SV40 promoter was transient and low, whereas expression driven by the Rous sarcoma virus (RSV) promoter was high and sustained, such that a 1,000-fold difference in expression could be observed. The mechanism of gene uptake is still unknown, but our findings indicate that fibers damaged by the injection procedure do not take up and express plasmid DNA.
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PMID:Direct gene transfer into skeletal muscle in vivo: factors affecting efficiency of transfer and stability of expression. 849 24

Sindbis virus was used as a self-amplifying eukaryotic expression vector. A recombinant cDNA genome of this (+)-strand RNA virus was placed under the transcriptional control of a Rous sarcoma virus LTR (RSV) promoter. Transfection of this plasmid construct into mammalian cell lines (3T3, HepG2, and 293 cells) resulted in expression of the luciferase reporter gene. High-expression levels were also measured after transfection into primary rat myoblasts. In differentiated myotubes, expression levels generated by the Sindbis virus vector were up to 200 times higher than those obtained with a conventional RSV expression vector. In vivo expression was detected after injection of plasmid DNA into mouse quadriceps. In vivo expression was transient and undetectable by day 16. This self-amplifying expression vector can be used for generating high-level expression of transgenes in vitro and in vivo. Its transient nature in vivo could allow for safe, short-term delivery of gene products in gene therapy protocols. It should facilitate the study of Sindbis and other RNA viruses.
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PMID:A plasmid-based self-amplifying Sindbis virus vector. 852 74

Fibroblast growth factors (FGFs) are important regulators of calvarial osteoblast growth and differentiation. We have studied the regulation of the osteoblast-specific gene osteocalcin (OC) by FGF2 in phenotypically immature MC3T3-E1 calvarial osteoblastic cells. FGF2 markedly induces OC mRNA accumulation in MC3T3-E1 cells in the presence of forskolin (FSK). Similarly, OC promoter activity (luciferase reporter) is up-regulated 6-10-fold by FGF2/FSK or by FGF2/8-bromo cyclic AMP. Half-maximal induction of OC promoter activity occurs at 1 nM FGF2. By 5' deletion analysis and dinucleotide point mutations, we map one component of this FGF2/FSK response to a GCAGTCA motif in the region -144 to -138 relative to the OC transcription initiation site. The OC promoter region -154 to -90 confers FGF2/FSK responsiveness on the Rous sarcoma virus minimal promoter. By 3' and internal deletion analyses, the region between -90 to -99 is also found to be necessary for FGF2/FSK synergy (encodes a PuGGTCA motif previously identified as a component of FSK induction). A DNA binding activity that recognizes the region -148 to -125 of the rat OC promoter is induced in crude nuclear extracts from MC3T3-E1 cells treated with FGF2 or FGF2/FSK. This binding activity is sequence-specific and does not recognize the TCAGTCA DNA cognate of AP1. Members of the ATF, Fos, and Jun family are not immunologically detected in this inducible DNA binding activity. However, transient co-expression of ATF3 but not ATF2 selectively attenuates the FGF2 component of induction. Thus, a novel FGF2-regulated DNA-protein interaction in the OC promoter participates in the transcriptional control of OC expression by FGF and cyclic AMP in MC3T3-E1 calvarial osteoblasts.
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PMID:Synergistic induction of osteocalcin gene expression: identification of a bipartite element conferring fibroblast growth factor 2 and cyclic AMP responsiveness in the rat osteocalcin promoter. 863 81

The A20 gene product is a novel zinc finger protein originally described as a tumor necrosis factor alpha (TNF)-inducible early response gene in human umbilical vein endothelial cells (HUVEC). Its described function is to block TNF-induced apoptosis in fibroblasts and B lymphocytes, but more recently it has also been shown to play a role in lymphoid cell maturation. The mechanism of action of A20 is unknown. The aim of our study was to assess the effect of A20 upon endothelial cell activation. By transfecting bovine aortic endothelial cells (BAEC) with A20 as well as reporter constructs consisting of the promoters of genes known to be up-regulated during endothelial cell activation, i.e. E-selectin, interleukin (IL)-8, tissue factor (TF), and inhibitor of nuclear factor kappaBalpha (IkappaBalpha), we demonstrate that A20 expression inhibits gene up-regulation associated with TNF, lipopolysaccharide (LPS), phorbol 12-myristate 13-acetate (PMA), and hydrogen peroxide (H2O2)-induced endothelial cell (EC) activation. The mechanism of action of A20 is in part, or totally, due to the blockade of nuclear factor kappaB (NF-kappaB), as shown by its ability to suppress the activity of a NF-kappaB reporter. This effect is specific, as A20 does not block a noninducible, constitutively expressed reporter, Rous sarcoma virus-luciferase (RSV-LUC); nor does it block the c-Tat-inducible, NF-kappaB-independent reporter, human immunodeficiency virus-chloramphenicol acetyltransferase (HIV-CAT). How A20 blocks NF-kappaB is unclear, although we demonstrate that it does not affect p65 (RelA)-mediated gene transactivation. The inhibition of endothelial cell activation by A20 is a novel function for A20.
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PMID:A20 blocks endothelial cell activation through a NF-kappaB-dependent mechanism. 866 99

A non-contracting scar following myocardial infarction can adversely affect ventricular topography and hemodynamic function. Gene transfer has the potential to prevent or alter such pathophysiological processes. Normal myocardium is a proven target for delivery of DNA or viral vectors but the potential for gene therapy in ischemic myocardium has not been evaluated. In an initial series of experiments, we determined whether the direct injection of reporter genes into hearts subjected to coronary artery occlusion followed by reperfusion could result in gene expression comparable to the levels observed in non-occluded normal hearts. Anesthetized rats were subjected to 15 min or 60 min of proximal coronary occlusion or sham operation. Luciferase gene under the control of the Rous sarcoma virus promoter was injected directly into the anterior left wall. At 1 week, high expression of luciferase was observed in both the ischemic/reperfused and non-ischemic tissue. Thus DNA transfer by direct injection is possible after ischemic injury and uptake and expression are not impaired. In a second series of experiments, myocardial infarcts in dogs were injected with a beta-galactosidase expressing retroviral vector. LNPOZ. Six to 11 days later frozen sections revealed macroscopically visible expression of beta-galactosidase activity. Not only can foreign genes be taken up by direct injection of DNA or retroviruses into ischemic/reperfused myocardium but they can be transcribed and the protein synthetic machinery of the injured cells can produce recombinant polypeptides that retain enzymatic activity. These results open the way for the investigation of gene therapy in models of ischemia.
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PMID:Ischemic/reperfused myocardium can express recombinant protein following direct DNA or retroviral injection. 874 21

We have designed and constructed a novel chimeric protein that consisted of a single domain of protein A and luciferase derived from sea-firefly Vargula hilgendorfii with the goal of obtaining a heterofunctional immunological tool. The structural gene of luciferase was fused to the 3' terminus of the D domain gene of protein A with/without a short linker of five amino acids. The resulting constructs under the transcriptional regulation of the Rous sarcoma virus (RSV) promoter, were expressed transiently in simian COS-1 and stably in Chinese hamster ovary (CHO) cells. The properties of the resultant chimeric protein were characterized. The results indicated that the dual properties of the chimeric protein could be retained only after the introduction of a linker of (Gly)4 Ser between the two conjugated moieties. Moreover, the chimeric protein was found to retain at least 50% of the specific activity as compared with the non-fused luciferase. The future prospect of the usage of this chimeric protein in the field of diagnostics was further evaluated by performing bioluminescent immunoassays.
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PMID:Expression of a bifunctional chimeric protein A-Vargula hilgendorfii luciferase in mammalian cells. 877 Apr 15

Adenoviruses are a promising vector system for future gene therapy of heart muscle diseases. The promiscuous tissue tropism of adenoviruses, however, may lead to the undesirable expression of putative therapeutic genes in nontarget cells and hence to considerable safety limitations for this vector system. To restrict gene expression to cardiomyocytes we constructed an adenoviral vector (Ad-mlcLuc) in which the luciferase gene is under the control of the ventricle-specific myosin light chain-2 (mlc-2v) promoter. For controls, we constructed a recombinant adenovirus without promoter (Ad-Luc) and one with the Rous sarcoma virus (RSV) promoter (Ad-rsvLuc). Our data demonstrate that the newly established viral vector Ad-mlcLuc was specifically active in rat neonatal cardiomyocytes in vitro but not in three established cell lines. Injections of the recombinant adenoviruses into the cardiac cavity of neonatal rats resulted in myocardial specific gene expression of Ad-mlcLuc in vivo, despite the fact that viral DNA was detected by PCR at different levels in all tissues investigated. In vitro and in vivo, Ad-mlcLuc was exclusively active in cardiac muscle cells, reaching 8-9% of the RSV-induced luciferase activity. Direct injection of Ad-mlcLuc into thigh muscle gave only background luciferase activity (0.05% of Ad-rsvLuc). Therefore, in the adenoviral system, the mlc-2v promoter allows heart-specific expression of a foreign gene thus providing a promising tool for gene transfer targeted to the myocardium.
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PMID:Heart muscle-specific gene expression using replication defective recombinant adenovirus. 890 6


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