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)

The bacterial gene for beta-galactosidase under the control of LTR from either human cytomegalovirus (CMV-lacZ) or the Rous sarcoma virus (RSV-lacZ) was injected into fertilized eggs of Misgurnus fossilis L. loaches and F1 hybrid mice (CBA x C57Bl). Expression of the CMV-lacZ was observed in almost 100% of the loach embryos and larvae for two months following the first day of embryonic development. In some cases, expression points were located only on either the right or the left side of a fish. The spectrum of tissues expressing CMV-lacZ was decreased during embryonic development: CMV-lacZ was expressed only in fin and body muscles of 6- to 8-week-old loaches. In mice, the RSV-lacZ gene was expressed in ectoderm- and mesoderm-derived tissues of a 13-day-old embryo, and the CMV-lacZ gene was expressed in tissues derived from various blastophylli of a 14-day-old embryo. Distribution of transgene expression is discussed with regard to authors' and published data.
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PMID:[Expression of the CMV-lacZ- and RSV-lacZ-genes in transgenic fish and mouse embryos]. 910 56

We have compared the in vitro and in vivo behaviors of a set of isogenic E1- and E1/E4-defective adenoviruses expressing the lacZ gene of Escherichia coli from the Rous sarcoma virus long terminal repeat. Infection of tumor-derived established cell lines of human origin with the doubly defective adenoviruses resulted in (i) a lower replication of the viral backbone that correlated with reduced levels of E2A-specific RNA and protein, (ii) a significant shutoff of late gene and protein expression, and (iii) no apparent virus-induced cytotoxicity. Independently of the extent of the deletion, the additional inactivation of E4 from the viral backbone therefore drastically disabled the virus in vitro, with no apparent effect on transgene expression. A lacZ-transgenic model was used to compare the different recombinant adenoviruses in the livers of C57BL/6 mice. The immune response to the virally encoded beta-galactosidase was minimal in this model, as infusion of the E1-defective adenovirus resulted in a time course of transgene expression that mimicked that in immunodeficient (nu/nu) mice, with very little inflammation and necrosis in the liver. Administration of a doubly defective adenovirus to the transgenic animals led to long-term extrachromosomal persistence of viral DNA in the liver, with no detectable methylation of CpG dinucleotides. However, transient transgene expression was observed independently of the extent of the E4 deletion, suggesting that the choice of the promoter may be critical to maintain transgene expression from these attenuated adenovirus vectors.
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PMID:Long-term gene delivery into the livers of immunocompetent mice with E1/E4-defective adenoviruses. 915 56

Adenoviruses are very attractive vectors for gene transfer into the cardiac muscle; however, their promiscuous tissue tropism, leading to an ectopic expression of the transgene, is a considerable practical limitation. To restrict expression of a reporter gene in cultured cardiomyocytes and in the heart of the rat, we have constructed a recombinant adenovirus (Ad-MLC2 beta gal) containing the beta-galactosidase gene under the control of the rat ventricle-specific cardiac myosin light chain 2 (MLC-2v) promoter. We show in this work that the MLC-2v promoter inside the adenoviral genome retains its cardiac specificity in vitro in cultured cardiomyocytes as well as in vivo in the animal heart. Northern blot studies after Ad-MLC2 beta gal infection show significant transcription only in cells derived from the cardiac muscle and not from the skeletal muscle. Quantitative analysis of the beta-galactosidase activity in a number of cell lines also confirms this result. The level of beta-galactosidase expression in rat neonatal cardiomyocytes infected with Ad-MLC2 beta gal is 8% of that found when primary cells are infected with Ad-RSV beta gal (containing a beta-galactosidase gene under the control of the Rous sarcoma virus promoter). The cardiomyocytes-specific expression is also found after injection of Ad-MLC2 beta gal directly into the rat myocardium, although the viral genome can be detected by polymerase chain reaction (PCR) in other tissues. Lack of expression after direct injection into liver and skeletal muscle confirms these results. The use of a tissue-specific promoter is a first step to restrict transgene expression to a particular cell type of the targeted tissue.
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PMID:Expression from cardiomyocyte-specific promoter after adenovirus-mediated gene transfer in vitro and in vivo. 918 Nov 18

The tissue kallikrein-kinin system has been postulated to play a role in blood pressure homeostasis and the pathogenesis of clinical hypertension. To demonstrate the potential therapeutic effects of somatic gene delivery in treating hypertension, we used spontaneously hypertensive rats (SHR) as a model. The gene encoding the human tissue kallikrein was used because of its powerful hypotensive action. The human kallikrein DNA constructs were placed under the control of the metallothionein metal response element, the cytomegalovirus promoter/enhancer or the Rous sarcoma virus 3'-LTR. The human tissue kallikrein DNA constructs were incorporated into adenoviral vectors via homologous recombination. The naked plasmid DNA constructs or adenovirus containing the kallikrein gene were first introduced into kidney 293 cells and the expression of human tissue kallikrein was identified by ELISA. The kallikrein gene was delivered into SHR via intramuscular, intravenous, portal vein, intraperitoneal, and intracerebroventricular routes. A single injection of naked human kallikrein DNA constructs caused a prolonged reduction of high blood pressure for up to 8 weeks. Adenoviral-mediated gene delivery results in high efficiency of human tissue kallikrein expression. Immunoreactive human kallikrein was detected in rat serum at the highest level at 1 day post gene delivery. Portal vein delivery of a reporter gene, AdCMV-LacZ, results in intense staining of beta-galactosidase in rat liver, suggesting that recombinant kallikrein is mainly produced in liver and secreted into the circulation. These results show that kallikrein gene delivery causes a sustained reduction of blood pressure in genetically hypertensive rats and provide important information for a potential gene therapy approach to human hypertension and related diseases.
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PMID:Kallikrein gene therapy: a new strategy for hypertensive diseases. 922 51

Application of neurotrophic factors (NFs) to the cut stump of motor nerves of neonatal rats confers neuroprotection from trauma-induced neuronal death. To test whether motoneurons are capable of responding to endogenously produced NFs, facial motoneurons were genetically modified in vivo to express several NFs and then tested for their response to peripheral nerve damage. Replication-defective adenoviral vectors [Adv. Rous sarcoma virus (RSV)-nf] representing three families of NFs were constructed that carried genes for brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), glial cell-derived neurotrophic factor (GDNF), and nerve growth factor. Media from cultured cells transduced with Adv. RSV-nf contained NFs that supported the survival of cultured chick sensory neurons in the same manner as recombinant NF standards. When Adv.RSV-nf or an adenoviral vector containing the beta-galactosidase gene (Adv.RSV-beta-gal) were injected into the facial muscles of neonatal rats the vectors were retrogradely transported to the facial nucleus where the NFs or beta-gal were expressed. A fraction (approximately 10%) of the neurons were transduced as demonstrated by reverse transcriptase-PCR, histochemistry, and immunocytochemistry. In the case of Adv.RSV-BDNF, Adv.RSV-CNTF, and Adv.RSV-GDNF, a significant portion of the facial nucleus neurons was protected, 16.5, 18.2, and 53.3%, respectively, from death after axotomy, showing that neurons are capable of transporting the Adv. RSV-nf, expressing the recombinant NF genes, and responding to the NFs. In the case of Adv.RSV-GDNF, a greater number of facial nucleus motoneurons survived than were transduced, indicating that neighboring untransduced neurons were protected by the GDNF expressed by the transduced neurons by a paracrine mechanism.
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PMID:Targeted transduction of CNS neurons with adenoviral vectors carrying neurotrophic factor genes confers neuroprotection that exceeds the transduced population. 925 62

Proliferation and the expression of proliferation-associated genes are modulated by changing the serum concentration in the media of cultured cells. To determine if activity of the SV40 early promoter is modulated by serum, we examined the expression of SV40 early promoter driven marker genes in murine BALB/c 3T3T cells following serum deprivation or serum stimulation. SV40-promoter-regulated beta-galactosidase and chloramphenicol acetyl transferase genes were studied following either transient or stable transfection. The results show that serum deprivation of growing cells induces SV40 promoter activity while serum stimulation of quiescent G0 cells suppresses it. Kinetic analyses show a significant induction of the SV40 promoter activity during the first 2 days of serum deprivation which is maintained at a high level for 15 days. The induction of reporter gene expression by serum deprivation was selective for the SV40 early promoter because such an effect was not observed using the Rous sarcoma viral promoter. Nuclear run-off assays further show that the transcription controlled by the SV40 early promoter is approximately twofold greater in cells rendered quiescent by serum deprivation for 72 h than in growing cells cultured in medium containing serum. These results suggest that one reason SV40 T transformed cells commonly fail to undergo quiescence following serum deprivation is that the SV40 promoter is induced.
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PMID:Serum deprivation induces SV40 early promoter activity. 933 95

Replication-defective adenoviruses have received increasing attention as vectors for exogenous gene administration in a variety of experimental and pathological conditions. However, little information exists about their utility for in utero gene therapy, and no information exists concerning their efficacy for gene delivery during initial organogenesis in the mammalian embryo. To evaluate the feasibility of using these vectors for exogenous gene transduction during the initial stages of organogenesis in the mammal, we injected an adenovirus vector carrying the bacterial beta-galactosidase (lacZ) gene under the control of either the cytomegalovirus (CMV) promoter or the Rous sarcoma virus (RSV) long terminal repeat (LTR) into early, post-gastrulation, mouse embryos, and evaluated expression following 36-48 h in culture. These studies suggest that adenovirus-mediated gene delivery may provide an efficient method of gene transduction during critical developmental stages with no detectable adverse effects on normal development during early morphogenesis. In addition, the type of promoter used had a significant effect on the tissue distribution of gene expression.
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PMID:Adenovirus-mediated gene transfer during initial organogenesis in the mammalian embryo is promoter-dependent and tissue-specific. 942 36

Recent advances have enabled transfer of genes to various types of cells and tissues. The goals of the present study were to transfer genes to nodose sensory neurons using replication-deficient adenovirus vectors and to define the conditions needed to optimize the gene transfer. Neurons were dissociated from rat nodose ganglia and maintained in culture. Cultures were exposed for 30 min to vectors containing the beta-galactosidase gene lacZ driven by either the Rous sarcoma virus (RSV) or the cytomegalovirus (CMV) promoter. Cultures were fixed and treated with X-gal to evaluate lacZ expression 1-7 days after exposure to virus. Increasing concentrations of virus led to dose-related increases in the number of neurons expressing lacZ. LacZ was expressed in 8 +/- 2, 39 +/- 6, and 82 +/- 3% of neurons 1 day after exposure to 10(7), 10(8), and 10(9) pfu/ml of AdRSVlacZ, respectively (P < 0.05). The same doses of AdCMVlacZ led to expression in 41 +/- 9, 60 +/- 10, and 86 +/- 4% of neurons. Expression driven by the CMV promoter was essentially maximal within 1 day and remained stable for at least 7 days. In contrast, expression driven by the RSV promoter was less on day 1 but increased over time (1-7 days). There was no lacZ expression in vehicle-treated cultures and exposure to the adenovirus vectors did not adversely influence cell viability. Exposure of the neuronal cultures to an adenovirus vector containing the gene for green fluorescent protein (AdRSVgfp, 10(9) pfu/ml) enabled visualization of successful gene transfer in living neurons. The results indicate that gene transfer to cultured nodose neurons can be accomplished using adenovirus vectors. The expression of the transferred gene persists for at least 7 days, occurs more rapidly when expression is driven by the CMV compared with the RSV promoter, and occurs without adversely affecting cell viability.
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PMID:Adenovirus-mediated gene transfer to cultured nodose sensory neurons. 942 4

We examined the ability of the human surfactant protein B (SP-B) promoter to confer cell specificity of transgene expression in an adenoviral vector. Using similar replication-deficient adenoviruses (rAd), we compared lacZ reporter gene expression driven by the human SP-B promoter (rAd.SPBlacZ) with the ubiquitously expressed Rous sarcoma virus promoter (rAd.RSVlacZ). rAd.SPBlacZ expressed lacZ in H-441 and A549 lung epithelial cell lines and not in HeLa cells whereas rAd.RSVlacZ expressed in all three cell lines. In primary human fetal lung fibroblasts, beta-galactosidase activity from rAd.RSVlacZ transduction increased in a dose-dependent manner whereas activity from rAd.SPBlacZ remained low. In mixed cell cultures prepared from human fetal lung explants that contained fibroblasts and type II cells, X-Gal staining localized rAd.SPBlacZ expression to only type II cells whereas rAd.RSVlacZ expressed in both cell types. In 24-wk gestation human fetal tissue explants infected ex vivo, the RSV promoter directed lacZ expression in lung, trachea, heart, liver, and esophagus, whereas with the SP-B promoter lacZ was expressed only in lung, specifically in air space-lining cells. This specificity was maintained in vivo. lacZ expression was undetectable in lung and other tissues after intravenous administration of rAd.SPBlacZ whereas rAd.RSV-lacZ expressed primarily in liver. After intratracheal instillation of rAd.SPBlacZ into mice, X-Gal staining localized expression to type II and Clara cells. In contrast, rAd.RSVlacZ expressed in all pulmonary epithelial cell types. Our results indicate that the SP-B promoter may be useful in targeting type II and Clara cells for gene therapy of conditions such as inherited deficiency of SP-B.
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PMID:Targeting type II and Clara cells for adenovirus-mediated gene transfer using the surfactant protein B promoter. 944 40

DNA plasmids formed particulate complexes with a variety of cationic polyamino acids and cationic lipids, which were used to transfect mammalian cells in culture. Complexation was studied by assaying for exclusion of ethidium using a fluorometric assay, which indicated that complexation with cationic polyamino acids took place with utilisation of the majority of charged functional groups. The particle sizes and zeta potentials of a range of complexes were determined. Generally polyamino acids formed uniform particles 80-120 nm in diameter in water, but their particle size increased on dilution of the particles in electrolytes or cell culture media. The efficiency of transfection was compared using complexes of pRSVlacZ, a reporter construct which expressed beta-galactosidase under the control of the Rous sarcoma virus promoter. Positively charged DNA/polyamino acid complexes were taken up by cells but required an endosomolytic agent, such as chloroquine, to facilitate transfection. Polyornithine complexes resulted in the highest levels of expression, in comparison with other homopolyamino acids (polyornithine>poly-L-lysine=poly-D-lysine>polyarginine). Copolyamino acids of lysine and alanine condensed DNA but were less active in transfection experiments. Copoly(L-Lys, L-Ala 1:1) was inactive even in the presence of chloroquine. In contrast DNA/cationic lipid complexes transfected cells spontaneously, and chloroquine did not improve the extent of expression, rather it usually reduced efficiency. There was little correlation between comparative efficiencies of lipid complexes between cell lines suggesting that the nature of the cell membrane and differences in mechanisms of internalisation were determinants of efficiency. In an effort to explore better cell culture models for gene delivery, monolayers of Caco-2 cells were transfected in filter culture. As the cells differentiated and formed a polarized monolayer, expression of beta-galactosidase was reduced until at day 27 expression was not significantly different from basal activity. The Caco-2 filter culture model merits further attention as a model of gene delivery to epithelial surfaces, such as would be encountered in the lung after inhalation.
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PMID:Polycation-DNA complexes for gene delivery: a comparison of the biopharmaceutical properties of cationic polypeptides and cationic lipids. 974 37


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