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)

Previous studies have demonstrated that a defective recombinant adenovirus can infect a wide range of postmitotic and slowly proliferating cell types such as hepatocytes, myotubes, pneumocytes and intestinal cells (Stratford-Perricaudet et al., Hum. Gene Ther., 1, 241-256, 1990; Quantin et al., Proc. Natl. Acad. Sci. USA, 89, 2581-2584, 1992; Jaffe et al., Nature Genetics, 1, 372-378, 1992). We have used a defective recombinant adenovirus, Ad.RSV beta gal, containing the Escherichia coli beta-galactosidase gene targeted to the nucleus under the transcriptional control of the Rous sarcoma virus long terminal repeat promoter (Stratford-Perricaudet et al., J. Clin. Invest., 90, 626-630, 1992) to infect non-dividing neural cells in primary culture. We show that 80-100% of neuronal and astroglial cells infected with a viral titre lower than 10(9) p.f.u./ml express beta-galactosidase for at least 1 month without cell damage. These results demonstrate the potential usefulness of recombinant adenovirus infection for the analysis of brain-specific gene regulation and for the transfer of genes into neural cells before their transplantation into the brain.
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PMID:Adenoviral vector as a gene delivery system into cultured rat neuronal and glial cells. 827 31

The formation of transgenic chimeric chickens for use in developmental studies and as intermediates in the production of transgenic chickens requires the incorporation of stably transfected blastodermal cells into a chimera. To obtain blastodermal cells, area pellucidae of stage X (Eyal-Giladi and Kochav, Dev. Biol. 49:321-337, 1976:E.-G.&K.) embryos were collected from unincubated, freshly oviposited Barred Plymouth Rock eggs and dissociated in 0.25% trypsin/0.04% EDTA (w/v) and 2% (v/v) chicken serum in phosphate-buffered saline (Ca2+ and Mg2+ free) at 4 degrees C for 10 min. The blastodermal cells were suspended in Dulbecco's Modified Eagle's Medium (DMEM) and transfected by lipofection with superhelical pmiwZ, a plasmid containing a hybrid lacZ gene encoding bacterial beta-galactosidase (beta-gal) under the control of a chicken beta-actin/Rous sarcoma virus promoter. A mixture of 2.5 micrograms Lipofectin and 1.56 micrograms pmiwZ in 250 microliters DMEM was incubated for 30 min at 37 degrees C and added to 500 microliters of 20-40,000 cells in suspension. Cells incubated with the transfection reagents in the presence or absence of pmiwZ were either plated and cultured for 48 h at 37 degrees C in 5% CO2/95% air, or injected through a shell window into the subgerminal cavity of White Leghorn stage X (E.-G.&K.) embryos previously exposed to 500-600 rads from a 60Co source, after which the window was sealed and the egg incubated at 38 +/- 1 degrees C for 72 h.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Efficient incorporation of transfected blastodermal cells into chimeric chicken embryos. 829 32

Control of Marek's disease in the poultry industry has been successfully achieved for several decades by large-scale vaccination of day-old chickens with live herpesvirus of turkeys (HVT) strains. Several features of this virus including lack of pathogenicity and long-term immune protection due to a persistent viraemic infection made us decide to use HVT as a live viral vector for the expression of foreign antigens. Potential sites for the integration of foreign DNA in the unique short region of the HVT genome were identified by the insertion of a beta-galactosidase expression cassette. Vaccination trials with recombinant virus strains indicated that the marker gene was expressed and stably maintained during animal passage. Based on an insertion site mapping in one of the open reading frames of the unique short region, a general recombination vector was designed for the integration of foreign genes into HVT. Recombinant virus-directed expression of individual antigens from Newcastle disease virus was driven by a strong promoter element derived from the lung terminal repeat sequence of Rous sarcoma virus.
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PMID:Avian herpesvirus as a live viral vector for the expression of heterologous antigens. 838 85

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

CCAAT/enhancer-binding protein (C/EBP) isoforms are thought to be important regulators of the hepatocyte phenotype. However, the specific physiological roles of different isoforms are poorly understood because hepatocytes express multiple C/EBPs, and various isoforms have overlapping functions. To identify the functions of C/EBPalpha in mature hepatocytes, replication-defective adenovirus vectors were used to efficiently and homogeneously overexpress the mouse C/EBPalpha gene in a SV40 virus-conditionally transformed rat hepatocyte line that can be induced to express C/EBPbeta and C/EBPdelta but that has little endogenous C/EBPalpha expression. Hepatocytes were infected with a recombinant adenovirus vector carrying the cDNA for C/EBPalpha driven by Rous sarcoma virus promoter elements (AdCEBPalpha) or a similar vector carrying the Escherichia coli lacZ gene (Adbetagal). Staining for beta-galactosidase demonstrated an infection efficiency of 100% at a multiplicity of infection of 25 plaque-forming units/cell and persistence of foreign gene expression for at least 9 days. Cultures infected with AdCEBPalpha had 50-fold higher levels of C/EBPalpha mRNA and protein than those infected with Ad-beta-gal, but similar expression of C/EBP-beta. Infection with AdCEBPalpha inhibited proliferation in cells expressing little C/EBPbeta, even when proliferation was driven by the SV40 transforming antigen, and also blunted mitogenic induction of the c-myc proto-oncogene in nontransformed cells with high levels of C/EBPbeta. Although overexpression of C/EBPalpha consistently increased C/EBPalpha DNA binding activity, it was not sufficient for albumin expression. Infection with AdCEBPalpha only increased albumin mRNA levels in nontransformed cells that also expressed relatively high levels of C/EBPbeta. Thus, in hepatocytes, C/EBPalpha has a dominant antiproliferative function, but must interact with other factors to regulate hepatocyte-specific gene expression.
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PMID:Adenovirus-mediated transfer of CCAAT/enhancer-binding protein-alpha identifies a dominant antiproliferative role for this isoform in hepatocytes. 863 55

This paper describes the use of the baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) as a vector for gene delivery into mammalian cells. A modified AcMNPV virus was prepared that carried the Escherichia coli lacZ reporter gene under control of the Rous sarcoma virus promoter and mammalian RNA processing signals. This modified baculovirus was then used to infect a variety of mammalian cell lines. After infection of the human liver cell lines HepG2, >25% of the cells showed high-level expression of the transduced gene. Over 70% of the cells in primary cultures of rat hepatocytes showed expression of beta-galactosidase after exposure to the virus. Cell lines from other tissues showed less or no expression of lacZ after exposure to the virus. The block to expression in less susceptible cells does not appear to result from the ability to be internalized by the target cell but rather by events subsequent to viral entry. The onset of lacZ expression occurred within 6 hr of infection in HepG2 cells and peaked 12-24 hr postinfection. Because AcMNPV is able to replicate only in insect hosts, is able to carry large (>15 kb) inserts, and is a highly effective gene delivery vehicle for primary cultures of hepatocytes, AcMNPV may be a useful vector for genetic manipulation of liver cells.
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PMID:Baculovirus-mediated gene transfer into mammalian cells. 863 76

In order to study transcriptional regulation of hepatic genes during development, a method for transfer of fusion genes to primary cultures of fetal hepatocytes was required. The aim of this study was to assess currently available transfection methods and optimize the best method for use with cultured fetal hepatocytes. The Rous sarcoma virus 5' long terminal repeat controlling transcription of the beta-galactosidase reporter gene (pRSV lac Z II) was used to assess electroporation, lipofection, DEAE-dextran and calcium phosphate transfection in cultured primary fetal hepatocytes. The success of transfection was determined by histochemical detection and quantitation of beta-galactosidase activity. Results showed that calcium phosphate transfection was optimal for fetal hepatocytes with respect to beta-galactosidase activity and cell survival. For maximum transfection of cells, 10 micrograms/ml DNA, HEPES buffered saline transfection buffer at pH 7.05 and a 24 hr expression period for the reporter gene were employed. Glycerol shock did not increase transfection efficiency significantly. The method was simplified by adding calcium chloride solution to DNA diluted in transfection buffer and the resulting co-precipitate added directly to the medium covering the cells. Transfection 24 hr after initial culture and a precipitate incubation time of 20 hr were optimal. The suitability of this method was confirmed with a liver-specific promoter controlling beta-galactosidase and chloramphenicol acetyltransferase expression. In conclusion this study shows that a modified calcium phosphate transfection method is most effective for transferring DNA to primary cultured fetal hepatocytes. It is concluded that this method is appropriate for use with fetal hepatocytes and will facilitate studies of gene regulation during liver development.
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PMID:Calcium phosphate transfection and cell-specific expression of heterologous genes in primary fetal rat hepatocytes. 867 28

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

Primary cultures of airway epithelia were used to evaluate variables pertinent to adenovirus (Ad)-mediated gene transfer efficiency and efficacy including: (i) Ad-vectors with different promoters, (ii) the duration of vector incubation with cells, (iii) the concentration and depth of vector-containing medium at constant multiplicity of infection (moi) (10(3)), and (iv) the relative sensitivity of reverse transcription polymerase chain reaction (RT-PCR) versus functional analysis for the detection of transduced cystic fibrosis transmembrane conductance regulator (CFTR). An Ad5-lacZ vector with a cytomegalovirus (CMV) enhancer/promoter transduced the greatest amount of beta-galactosidase (beta-Gal) activity, while an Ad2-lacZ vector with an E1a enhancer/promoter transduced the least. Ad5-lacZ vectors with the Rous sarcoma virus (RSV), E1a/RSV, or CMV enhancer/beta-actin (CB) promoters transduced intermediate levels of beta-Gal. Optimal gene transfer efficiency was detected with a 4-8 hr incubation of Ad5-CMVlacZ with cells, although optimal CFTR Cl-transport function was detectable after only a 30 min incubation of Ad5-CBCFTR with cells, consistent with correction of > or = 6-10% of cells in the epithelial sheet. Ad5-CBCFTR transduction of CF airway epithelial cells (moi = 10(3)) was optimal when higher concentrations, lower volumes, or smaller depths of vector-containing medium were utilized. RT-PCR was at least 100-fold more sensitive for the detection of transduced CFTR than functional analysis, and could detect as few as 0.001% Ad5-CBCFTR-infected CF cells admixed with uninfected CF cells. In summary, the variables studied clearly affect the efficiency of Ad-mediated gene transfer in vitro and potentially in vivo. They also suggest that RT-PCR is a poor marker of gene transfer efficiency and efficacy.
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PMID:In vitro assessment of variables affecting the efficiency and efficacy of adenovirus-mediated gene transfer to cystic fibrosis airway epithelia. 882 68

Myocardial infarcts heal by scar formation because there are no stem cells in myocardium, and because adult myocytes cannot divide and repopulate the wound. We sought to redirect the heart to form skeletal muscle instead of scar by transferring the myogenic determination gene, MyoD, into cardiac granulation (wound repair) tissue. A replication-defective adenovirus was constructed containing MyoD under transcriptional control of the Rous sarcoma virus long terminal repeat. The virus converted cultured cardiac fibroblasts to skeletal muscle, indicated by expression of myogenin and skeletal myosin heavy chains (MHCs). To determine if MyoD could induce muscle differentiation in vivo, we injected 2 x 10(9) or 10(10) pfu of either the MyoD or a control beta-galactosidase adenovirus into healing rat hearts, injured 1 wk previously by freeze-thaw. After receiving the lower viral dose, cardiac granulation tissue expressed MyoD mRNA and protein, but did not express myogenin or skeletal MHC. When the higher dose of virus was administered, double immunostaining showed that cells in reparative tissue expressed both myogenin and embryonic skeletal MHC. No muscle differentiation occurred after beta-galactosidase transfection. Thus, MyoD gene transfer can induce skeletal muscle differentiation in healing heart lesions. Modifications of this strategy might eventually provide new contractile tissue to repair myocardial infarcts.
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PMID:Muscle differentiation during repair of myocardial necrosis in rats via gene transfer with MyoD. 894 36


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