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)

Monocyte/macrophage cell lines are fastidious cells commonly used in transient transfection experiments. In the course of a study of gene regulation by lipopolysaccharide (LPS), we have compared several methods for DNA-mediated cell transfection to determine which would be optimally applicable to the macrophage line, RAW 264.7. Both the response level (LPS inducibility) and the degree of inter-assay variation were evaluated for each transfection technique. The following methods were compared: Lipofectin, LipofectAMINE, LipofectAMINE PLUS, SuperFect, Ca3(PO4)2 DNA co-precipitation, DEAE dextran-mediated transfection and electroporation. The transfected plasmid DNA included a luciferase reporter construct containing the junB minimal promoter under the control of an LPS-inducible 1300-bp regulatory fragment downstream of junB 5'-flanking sequence, as well as a beta-galactosidase reporter construct under the adenovirus promoter and enhancer used as an internal control. Electroporation, followed by a resting period of 16-24 h before stimulation with LPS, had the highest inducibility of all methods. DEAE dextran and Ca3(PO4)2 precipitation showed the least and the greatest inter-assay variation, respectively. For all other methods, inter-assay variability fell within this range. The results presented may serve as both a general reference and a guide for reporter gene studies in this or other macrophage cell lines.
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PMID:Evaluation of methods for transient transfection of a murine macrophage cell line, RAW 264.7. 1052 25

The role of viral immediate-early (IE) gene expression in herpes simplex virus type 1 (HSV-1) latency was investigated. The HSV-1 multiple mutant in1312, defective for the expression of the virion transactivator VP16 and the IE proteins ICP0 and ICP4, was used as the parent for these studies. The coding sequences of the Escherichia coli lacZ gene, preceded by the encephalomyocarditis virus internal ribosome entry site, were inserted into the region of in1312 that encodes the latency-associated transcripts (LATs) such that transcription of the transgene was controlled by the LAT promoter. This insert has previously been shown to direct long-term latent-phase expression of beta-galactosidase in a wild-type HSV-1 genome (R. H. Lachmann and S. Efstathiou, J. Virol. 71, 3197-3207, 1997). The resulting recombinant, in1388, was apathogenic after inoculation into mice via the footpad and did not detectably replicate in dorsal root ganglia (DRG) or footpads. Mutant in1388 established latency in DRG, and beta-galactosidase was expressed in increasing numbers of neurons over the first 25 days of infection. During latency, more than 1% of neurons in ganglia that innervate the footpad expressed beta-galactosidase, with the number of positive cells remaining constant for at least 5 months. Rescue of the VP16, ICP0, or ICP4 mutations of in1388 did not affect the number of beta-galactosidase-expressing neurons detected during latency. The results demonstrate that HSV-1 mutants severely impaired for IE gene expression are capable of establishing latency and efficiently expressing a foreign gene product under control of the LAT promoter.
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PMID:Long-term transgene expression in mice infected with a herpes simplex virus type 1 mutant severely impaired for immediate-early gene expression. 1062 58

Experimental investigation of glioma biology and therapy requires a representative model and a convenient technique for regulating gene expression. We have established an in vivo model in which genetically modified rat C6 glioma cells (C6TL cells) are transplanted into nude mice brain, followed by specific transcriptional control of a transgene. Histologically, the tumors exhibit an astrocytic phenotype and closely resemble human malignant gliomas including diffuse brain invasion. Due to a stably integrated lacZ gene, individual tumor cells can be unequivocally identified in tissue sections by histochemistry for beta-galactosidase. Since C6TL cells carry the tet transactivator (tTA) gene, any additional gene under control of a tetracycline/tTA-responsive promoter can be transcriptionally regulated by the concentration of tetracycline. C6TL cells stably transfected with a tetracycline/tTA-responsive luciferase reporter gene showed 23-fold regulation of luciferase activity in vitro. After intracerebral transplantation a regulation of 4.5- to 8.3-fold was obtained, dependent on the concentration and the type of tetracycline in the drinking water. This model should be useful for studying the functional role of candidate genes in tumor biology as well as for experimental gene therapy studies.
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PMID:In vivo glioma model enabling regulated gene expression. 1086 92

To produce conditional expression of genes in the mouse epidermis we have generated transgenic mouse lines in which the tetracycline-regulated transcriptional transactivators, tTA and rTA, are linked to the bovine keratin 5 promoter. The transactivator lines were crossed with the tetOlacZ indicator line to test for transactivation in vivo. In the absence of doxycycline, the K5/tTA line induced beta-galactosidase enzyme activity in the epidermis at a level 500-fold higher than controls, and oral and topical doxycycline caused a dose- and time-dependent suppression of beta-galactosidase mRNA levels and enzyme activity. In the K5/rTA lines, doxycycline induced beta-galactosidase activity between 3- and 50-fold higher depending on the founder line, and this occurred within 24-48 h after dosing. Histochemical analysis of all lines localized beta-galactosidase expression to the basal layer of the epidermis and the outer root sheath of the hair follicle, as well as other keratin 5 positive tissues. In several K5/rTA lines, skin-specific transactivation was restricted to the hair follicle. Treatment of these double transgenic mice with 12-O-tetradecanoyl-phorbol-13-acetate caused rapid migration of beta-galactosidase marked cells from the hair follicle through the interfollicular epidermis, demonstrating the usefulness of this specific double transgenic for fate mapping cells in the epidermis. These results show that the tetracycline regulatory system produces effective conditional gene expression in the mouse epidermis, and suggest that it should be amenable to suppression and activation of foreign genes during development and specific pathologic conditions relevant to the epidermis.
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PMID:Conditional gene expression in the epidermis of transgenic mice using the tetracycline-regulated transactivators tTA and rTA linked to the keratin 5 promoter. 1106 15

The ability to control gene expression in a temporal and spatial manner provides a new tool for the study of mammalian gene function particularly during development and oncogenesis. In this study the suitability of the tet-system for investigating embryogenesis was tested in detail. The tTACMV(M1) and rTACMV-3 (reverse Tc-controlled transactivator) transgenic mice were bred with NZL-2 bi-reporter mice containing the vector with a tTA/rTA responsive bidirectional promoter that allows simultaneous regulation of expression of two reporter genes encoding luciferase and beta-galactosidase. In both cases reporter genes were found to be expressed in a wide spectrum of tissues of double transgenic embryos and adult mice. The earliest expression was detected in tTACMV(M1)/NZL-2 embryos at embryonic day 10.5 (E10.5) and rTACMV-3/NZL-2 embryos at E13.5. Doxycycline abolished beta-gal expression in tTACMV(M1)/NZL-2 but induced it in rTACMV-3/NZL-2 embryos including late stages of embryo-genesis. The tTA and rtTA transactivators thus revealed a partially complementary mode of action during second half of embryonic development. These experiments demonstrated that both Tet regulatory systems function during embryonic development. We conclude that the Tet systems allows regulation of gene expression during embryonic development and that 'double reporter' animals like the NZL-2 mice are useful tools for the characterization of newly generated tet transactivator lines expressing tTA (or rtTA) in embryonic as well as in adult tissues.
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PMID:Tet-system for the regulation of gene expression during embryonic development. 1143 81

Despite the overall successful application of the tet-system to regulate gene expression in vitro and in vivo, nothing is known so far about the long-term stability of this system in transgenic mice. In this study, mice of generation F2, F3, F4, or F10 of two independent tTA(CMV) transgenic lines were bred with NZL-2 mice containing a tTA-responsive bidirectional promoter that allows the simultaneous expression of two reporter genes encoding luciferase and beta-galactosidase. Analysis of the expression of transgenes in double transgenic mice revealed a dramatic reduction of tTA transactivator mRNA over time. As a consequence, the expression of both reporter genes was gradually reduced from generation to generation in tissues of embryonic and adult NZL-2/tTA(CMV) mice. Luciferase activity in NZL-2/tTA(CMV)(F10) mice was reduced 8-10-fold compared to NZL-2/ tTA(CMV)(F2) mice, and beta-galactosidase expression was no longer detectable. In summary, we describe the long-term instability of the tet-system in our NZL-2/tTA(CMV) double transgenic mice. The molecular basis of this observation and experimental tools to overcome this limitation need to be addressed in future.
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PMID:Generation dependent reduction of tTA expression in double transgenic NZL-2/tTA(CMV) mice. 1166 82

Crx, an Otx-like homeobox gene, is expressed primarily in the photoreceptors of the retina and in the pinealocytes of the pineal gland. The CRX homeodomain protein is a transactivator of many photoreceptor/pineal-specific genes in vivo, such as rhodopsin and the cone opsins. Mutations in Crx are associated with the retinal diseases, cone-rod dystrophy-2, retinitis pigmentosa, and Leber's congenital amaurosis, which lead to loss of vision. We have generated transgenic mice, using 5'- and/or 3'-flanking sequences from the mouse Crx homeobox gene fused to the beta-galactosidase (lacZ) reporter gene, and we have investigated the promoter function of the cell-specific and developmentally regulated expression of Crx. All of the independent transgenic lines commonly showed lacZ expression in the photoreceptor cells of the retina and in the pinealocytes of the pineal gland. We characterized the transgenic lines in detail for cell-specific lacZ expression patterns by 5-bromo-4-chloro-3-indolyl beta-D-galactoside staining and lacZ immunostaining. The lacZ expression was observed in developing and developed photoreceptor cells. This observation was confirmed by coimmunostaining of dissociated retinal cells with the lacZ and opsin antibodies. The ontogeny analysis indicated that the lacZ expression completely agrees with a temporal expression pattern of Crx during retinal development. This study demonstrates that the mouse Crx 5'-upstream genomic sequence is capable of directing a cell-specific and developmentally regulated expression of Crx in photoreceptor cells.
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PMID:The mouse Crx 5'-upstream transgene sequence directs cell-specific and developmentally regulated expression in retinal photoreceptor cells. 1188 Apr 94

The late genes of the temperate phage of Pseudomonas aeruginosa are organized in an analogous fashion to the corresponding transcription units of the Escherichia coli P2 and P2-like phages. Sequence analysis of four putative late promoter regions, PP(phiCTX), PO(phiCTX), PV(phiCTX) and PF(phiCTX), reveals no similarity to sigma(70)-type promoters or promoter consensus sequences found in Pseudomonas, indicating the apparent need for a phage-encoded protein to control the expression of phiCTX late genes. To elucidate the mode of expression of the late genes, we fused the putative late promoter regions to the promoterless lacZalpha gene, which encodes the N-terminal part of beta-galactosidase as a reporter enzyme, in the promoter-probe vector pME4510. The candidate transactivator gene orf34 was cloned into expression vector pHA10, to generate the plasmid pHA34. The two recombinant plasmids were introduced together into E. coli XL1-Blue and P. aeruginosa PAO1S-Lac. Our results demonstrate that in phiCTX three late promoters (PP(phiCTX), PO(phiCTX), and PF(phiCTX)) are activated upon induction by IPTG in PAO1S-Lac carrying the cloned promoters and pHA34. Deletions and base-pair substitutions obtained by PCR-mediated mutagenesis demonstrated that two conserved sequences, TTGTAG-N(9)-cTACAa and GcCGCGCGCGCGgC, are essential for effective late gene expression. Whereas the late promoters were active in P. aeruginosa, only weak beta-galactosidase activity was obtained in E. coli.
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PMID:Organization and activation of the late promoters of phiCTX, a cytotoxin-converting phage from Pseudomonas aeruginosa. 1191 13

Regulated expression of therapeutic genes is required for long-term gene therapy applications for many disorders. Here we describe a doxycycline (dox)-regulated lentiviral vector system consisting of two HIV-1-based self-inactivating viruses. One of the vectors is constitutively expressing a novel improved version of the tetracycline reverse transactivator rtTA2(S)-M2 and the other has a rtTA responsive promoter driving the expression of beta-galactosidase gene (lacZ). The rtTA2(S)-M2 has highly improved properties with respect to specificity, stability and inducibility. Functionality of the system by dox was confirmed after in vitro cotransduction of Chinese hamster ovary and human endothelial hybridoma (EAhy926) cells. Regulation of the system showed tight control of the gene expression. Dose dependence for dox was seen with concentrations that can be obtained in vivo with doses normally used in clinical practice. LacZ expression could be switched on/off during long-term (3 months) culturing of cotransduced cells. The system was next tested in vivo after cotransduction into rat brain and studying expression of the lacZ gene in dox-treated and control rats. Nested RT-PCR confirmed that the tight control of the gene expression was achieved in vivo. Also, X-gal staining showed positive cells in the dox-treated rats, but not in the controls 10 days after cotransduction with 4 days preceding treatment with dox. It is concluded that our doxycycline-regulated vector system shows significant potential for long-term gene therapy treatments.
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PMID:Doxycycline-regulated lentiviral vector system with a novel reverse transactivator rtTA2S-M2 shows a tight control of gene expression in vitro and in vivo. 1262 50

Long-term constitutive secretion of insulin by implantation of ex vivo transfected cells such as fibroblasts or myoblasts or in situ by intramuscular injection of naked plasmid DNA provides a potential approach to gene therapy for diabetes mellitus. A mechanism for regulating insulin secretion will be necessary to realize the therapeutic potential of this approach. A second obstacle is the inability of non-endocrine host cells to fully process proinsulin. Therefore, alteration of the wild-type cDNA will be necessary to achieve processing of proinsulin by endogenous endoproteases within these cells. The cDNAs for beta-galactosidase (beta), human wild-type proinsulin (hppI1) and a mutated construct (hppI4), in which the dibasic PC2 and PC3 cleavage sites had been altered to form furin cleavage sites, were sub-cloned into four vectors (pCR3, pVR1012, pIRES, pTRE), including a tetracycline responsive plasmid (pTRE) that requires co-transfection with another plasmid encoding a transactivator (pTet-off) for transgene expression. Transient transfection of the COS-7 fibroblast cell line with these constructs was performed using DEAE-dextran and liposomes. Analysis of vector efficiencies revealed that pTRE/pTet-off>pIRES>pCR3>pVR1012. Further analysis demonstrated total pro/insulin secretion of 2.33 ng/10(6) cells/24 h with > or =25% processed to insulin in hppI-1.pTRE/pTet-off-transfected cells compared with 0.39 ng/10(6) cells/24 h and >70% processing in hppI-4.pTRE/pTet-off-transfected cells. In co-transfection studies with pTRE-hppI1/pTet-off and pTRE-hppI4/pTet-off constructs, pro/insulin secretion was inhibited to 65-66% and 36-38% of control (100%) in the presence of 0.01 and 0.1 microg/ml tetracycline respectively over a 24-h incubation period. Furthermore, reversal of tetracycline inhibition was demonstrated for pTRE-hppI1/pTet-off- and pTRE-hppI4/pTet-off-transfected cells. After a 48-h incubation with 1.0 microg/ml tetracycline, total pro/insulin levels were 10 and 14% compared with untreated cells respectively. On tetracycline removal, total proinsulin levels increased and were equivalent to untreated groups 72 h later. In conclusion, regulation of fully processed human insulin secretion has been achieved in a transiently transfected non-endocrine cell line.
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PMID:Tetracycline-regulated secretion of human insulin in a transfected non-endocrine cell line. 1279 Aug 3


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