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)

Recently, much attention has been paid to the arterial gene transfer technique using several vector systems. In the present study, we experimentally examined the transfection efficiency of the exogenous gene, the duration of gene expression, and the cytotoxic effect of the hemagglutinating virus of Japan (HVJ) liposome on the intact arterial wall to evaluate its effectiveness for the study of arterial diseases. To evaluate the transfection efficiency and duration of gene expression, pSV beta-galactosidase was transferred into the carotid arterial wall of rabbits. The cytotoxic effect of HVJ liposomes on the vascular cell components was also evaluated by a neutral red assay in vitro and scanning electron microscopy in vivo. HVJ liposomes could achieve highly efficient gene transfection into the medial smooth muscle cells of intact arteries at 150 and 760 mmHg of pressure (mean = 85.3% and 93.5% of total smooth muscle cells, respectively) without any inflammatory reaction. The introduced exogenous gene was expressed for at least 14 days. In addition, the cytotoxicity for the arterial smooth muscle cells and endothelial cells induced by HVJ liposome vehicles at routinely used concentrations (5,000 to 10,000 hemagglutinating activity units/ml) was minimal both in vitro and in vivo. As an example, we introduced human vascular endothelial growth factor cDNA, which was driven with cytomegalovirus enhancer and beta-actin promoter, into the rabbit carotid arteries, and it induced not only angiomatoid proliferation of endothelial cells forming irregular vascular channels but also intimal hyperplasia. Based on these findings, we conclude that HVJ liposome-mediated arterial gene transfer is a highly efficient, noninvasive, and effective gene delivery method for the study of vascular disorders.
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PMID:Characterization of in vivo gene transfer into the arterial wall mediated by the Sendai virus (hemagglutinating virus of Japan) liposomes: an effective tool for the in vivo study of arterial diseases. 880 55

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

For the purpose of studying the factors that cause wide variation in transient transgene expression in individual fish, a lacZ reporter gene linked to a carp beta-actin regulatory sequence was introduced into zebrafish embryos. As a general trend, a correlation between the number of transgene copies injected and the level of transgene expression was found. However, a substantial variation in the level of expression still occurred that could not be attributed to technical factors such as the difference in injected volume of the transgene. Co-injection of 32P-dCTP and transgene into the same embryo followed by detection of beta-galactosidase activity, has shown that the volume used for transgene injection, which was determined in terms of radioactivity, is not closely related to the level and location of transgene expression. Injection into the animal pole at zygote stage and the yolk cytoplasmic layer (YCL) at the 64-cell stage followed by determination of transgene expression in terms of unit injection volume, revealed that there are marked differences among tissues with regard to their capacity for transgene expression, and that the yolk syncytial layer is higher in this capacity. This high activity is assumed to be due to the high transcriptional activity or enhanced transgene replication in the syncytial layer, which is known to contain giant polyploid nuclei. The high levels of expression in the YSL may influence transient expression studies using quantitative comparative analyses and should be taken into consideration when expression data are derived from homogenates of yolk sac embryos.
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PMID:High transgene activity in the yolk syncytial layer affects quantitative transient expression assays in zebrafish Danio rerio) embryos. 884 May 26

A comparative study on the level of expression of lacZ reporter constructs driven by equivalent carp and rat beta-actin regulatory sequences was carried out in embryos of tilapia and rainbow trout. DNA was microinjected into fertilised tilapia and rainbow trout eggs and the embryos/fry were assayed at various developmental stages for beta-galactosidase expression. We provide evidence to demonstrate that the carp beta-actin promoter/ lacZ reporter gene is expressed at higher levels than the equivalent rat beta-actin construct in both species.
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PMID:Comparison of the activity of carp and rat beta-actin gene regulatory sequences in tilapia and rainbow trout embryos. 891 67

We evaluated the efficiency of gene transduction and of gene expression by adenoviral vectors in human lung adenocarcinoma cells. Freshly isolated cancer cells were collected from pleural effusions in adenocarcinoma patients by centrifugation with a Percoll gradient. Adenoviral vectors resulted in effective gene transduction into human lung cancer cell lines and into freshly isolated lung adenocarcinoma cells. In an experiment using the beta-galactosidase (LacZ) gene, the Adex1CA vector with a regulatory sequence of chicken beta-actin as promoter and an enhancer derived from cytomegalovirus produced a higher transduction ratio and greater expression levels than adenoviral vectors with other promoter systems. Transduction with Adex1CA vectors containing the human interleukin-2 (IL-2) gene (Adex1CAhIL-2) resulted in enhanced secretion of IL-2 from gene-modified lung cancer cells. Treatment with normal human serum inhibited gene transduction by Adex1CAhIL-2 but did not inhibit gene expression after transduction by Adex1CAhIL-2. The secretion of IL-2 from the gene-modified cells, which were irradiated at 100 Gy before transduction, continued for 8 days. In a mouse model, the intrapleural injection of IL-2 gene-modified 3LL cells transduced by Adex1CAhIL-2 could cure the pre-existing lung tumours with malignant pleural effusions to induce tumor-specific immunity. But these therapies did not show any therapeutic benefit on the pre-existing tumor in subcutaneous region. These data suggest a potentially useful but limited clinical role of Adex1CAhIL-2 in gene therapy for lung cancer patients.
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PMID:Interleukin-2 gene transduction into freshly isolated lung adenocarcinoma cells with adenoviral vectors. 898 90

An attempt was made to improve gene transfer into chick embryos in order to produce transgenic chickens. The beta-actin-lacZ/MiwZ, a marker gene in transfection reagent, was injected into the blastodisc of either unincubated fertilized eggs (stage X) or eggs induced from the shell gland by treating the hens intravenously with oxytocin or arginine vasotocin (stages IV-VI). All the manipulated embryos were incubated to reach stage XIV, the period at which primordial germ cells (PGCs) migrate from the germinal crescent to the gonadal anlage via the blood stream. MiwZ was detected in the embryos, extraembryonic tissues and blood by the histochemical staining method of beta-galactosidase. The MiwZ DNA was detected in 57% (127/221) of the survival embryos and in 9% (12/127) of the embryonic tissues. The expression was observed mosaically in the epidermis, heart and neural tube. The PGCs in the blood collected from the vitelline artery or dorsal aorta also showed a positive histochemical staining. However, the expression of MiwZ using the soft shelled eggs was more intense in the extraembryonic tissues, although it did not emerge in the embryos. Thus, it is possible to introduce an exogenous gene into the embryonic tissues using incubated fertilized eggs without sacrificing the hens. This technique for successive genetic operations should facilitate the production of transgenic chickens.
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PMID:In vivo gene transfer into the blastoderm of early developmental stage of chicken. 911 91

The mechanism which is responsible for the association of chronic hepatitis B virus (HBV) infection with hepatocellular carcinoma (HCC) is poorly understood. The protein encoded by the HBV X-gene (HBx) has been identified as potentially oncogenic. HBx is a promiscuous indirect trans-activator of a wide range of cellular and viral cis-elements and may disrupt the maintenance of genomic integrity by inhibiting p53 function and binding a putative DNA repair protein (XAP-1). In this report, we show that there is preferential binding of recombinant HBx to damaged DNA through an association with nuclear proteins. We have used the transcriptional activation by HBx of the beta-actin promoter of a beta-galactosidase reporter cassette to label cultured Chang liver cells expressing HBx. We demonstrate that cells expressing HBx are sensitised to the lethal effects of low dose ultraviolet irradiation. These data indicate that HBx interferes with liver cell DNA repair by binding damaged DNA and may predispose to the accumulation of potentially lethal or carcinogenic mutations.
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PMID:Hepatitis B virus X-protein binds damaged DNA and sensitizes liver cells to ultraviolet irradiation. 912 43

Recent reports have suggested that delivery of genes flanked by AAV ITRs may be useful for gene therapy of diseases that involve the brain. We have compared the efficiency of gene expression in vitro in CNS-derived cells from four different promoters when the transgene is flanked by AAV ITRs, using both transfection via cationic liposomes, and infection via rAAV. The human cytomegalovirus (CMV) immediate-early enhancer/promoter, the SV40 early enhancer/promoter, the JC polymovirus promoter, and the chicken beta-actin promoter coupled to the CMV enhancer were able to drive expression of the reporter gene beta-galactosidase in all tumor and primary brain cell cultures tested. Although the relative order of efficiency differed between cell types, the CMV promoter was always the strongest, generally by at least one order of magnitude. A comparison of the relative levels of expression seen between different cell types on transfection and infection suggest that not all CNS-derived cells are infected equally efficiently by rAAVs. High level of expression were seen within 24 h of transgene delivery by either transfection or infection, dropping dramatically within days. All cell types and promoters showed the same decline, suggesting that transient expression by rep-rAAVs may be efficient, but stable expression as detected in this system is a low frequency event. In vivo studies using the CMV promoter also suggest that although rep-rAAVs are able to infect efficiently CNS cells and produce high levels of gene expression shortly after transduction, the majority of such infections do not lead to stable high-level expression of transgenes.
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PMID:Comparison of promoter strengths on gene delivery into mammalian brain cells using AAV vectors. 915 5

The transient expression of reporter gene constructs in embryos provides a powerful tool to characterise cis-acting transcriptional elements of the genes involved in development. In the present study, we have analysed the expression pattern of several muscle-specific and ubiquitous regulatory sequences in microinjected zebrafish embryos. By using a fast and reproducible coinjection strategy, the mosaic expression of lacZ reporter gene was monitored in wholemount embryos injected with sequences containing putative enhancer elements and a carp myosin heavy chain promoter/lacZ reporter construct. We have found that a 0.9-kb myosin heavy chain (MyHC) proximal promoter containing several putative myogenic regulatory factors (MRF) binding sites is sufficient to restrict lacZ expression to the skeletal muscle fibres of prim-6 stage zebrafish embryos. Expression of a rat-derived foetal myosin light chain enhancer (MyLC) and different fragments of a carp beta-actin regulatory region together with the MyHC promoter were compared by accumulating the type, number and spatial distribution of beta-galactosidase-expressing cells on an expression map. beta-galactosidase activity increased similarly whether the MyLC enhancer was ligated to the promoter/ reporter construct directly or when coinjected as a separate fragment whilst skeletal muscle specificity was retained. The coinjection of two different forms of the beta-actin regulatory elements also showed a marked effect on the MyHC promoter activity. The coinjection of putative enhancers with minimal promoter constructs and subsequent analysis of the transient expression pattern in the developing embryos provides a rapid and simple technique to identify cis acting activator elements of genes expressed in the vertebrate embryo.
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PMID:Activator effect of coinjected enhancers on the muscle-specific expression of promoters in zebrafish embryos. 921 24

The Cre-loxP recombination system of bacteriophage P1 is frequently utilized in genetic manipulation in embryonic stem (ES) cells. The level of Cre expression is critical to induce loxP site-specific recombination in ES cells. To compare the efficiency of recombination, we constructed four Cre expression vectors driven by different promoters: cytomegarovirus/chicken beta-actin (CAG) promoter, human polypeptide chain elongation factor 1alpha (hEF-1alpha) promoter, mouse phosphoglycerate kinase-1 (mPGK) promoter, and polyoma enhancer/herpes simplex virus thymidine kinase (MC1) promoter. We introduced these Cre expression vectors by electroporation into three ES cell lines carrying a single copy of CAG-loxP-chloramphenicol acetyltransferase (CAT) gene-loxP-beta-galactosidase (beta-gal) gene construct. Since the Cre-mediated recombination leads to excision of the CAT gene, the efficiency of recombination can be monitored as beta-gal expression. No selection system was used in the experiments. The maximum recombination frequency was obtained when the CAG promoter was used, followed by the hEF-1alpha promoter, the mPGK promoter and the MC1 promoter in order. These results indicate that the efficiency of recombination in transient expression system correlates with the promoter activity of Cre expression vector. Thus, it is important to choose the promoter for effective recombination by Cre.
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PMID:Efficiency of recombination by Cre transient expression in embryonic stem cells: comparison of various promoters. 944 13


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