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At least 73 records · Page 4Linked to original sources
PubMed2000

A fragment of the Neurogenin1 gene confers regulated expression of a reporter gene in vitro and in vivo.

The basic helix-loop-helix transcription factor neurogenin1 is required for proper nervous system development in vertebrates. It is expressed in neuronal precursors during embryonic development and is thought to play a role in specifying neuronal fate. To investigate the regulation of neurogenin1 expression, the transcriptional start site of the gene was identified and a 2.7-kb fragment ending in the first exon was shown to possess basal promoter activity. This 2.7-kb fragment was able to promote expression of reporter genes in P19 cells under conditions in which expression of endogenous neurogenin1 was induced. Importantly, the 2.7-kb fragment was able to drive expression of a lacZ reporter gene in transgenic mice in most tissues in which neurogenin1 is normally expressed, including those peripheral ganglia that fail to develop in neurogenin1 "knockout" mice. These findings identify a regulatory region containing elements responsible for appropriate expression of a gene with a crucial role in generating the vertebrate nervous system.

Animals↗
PubMed1993

Regulatory sequences on the human villin gene trigger the expression of a reporter gene in a differentiating HT29 intestinal cell line.

To develop a molecular tool for tissue-specific targeting of gene expression in immature and differentiated epithelial cells of the small and large intestinal mucosa, we have isolated the 2-kb 5'-flanking region of the human villin gene. This region contains numerous short sequences that are conserved among other tissue-specific promoters of genes expressed in differentiated enterocytes. This DNA fragment promotes the transcription and expression of the luciferase reporter gene in villin-positive intestinal, renal, and hepatoma cell lines but not in a villin-negative keratinocyte cell line. The pattern of expression corresponds that of the endogenous gene, indicating that this sequence can direct intestine-specific transcription. In the differentiating HT29 intestinal cell line, expression of the reporter gene is already detectable in undifferentiated cells, and dramatically increases when terminal differentiation is induced. Thus, as previously reported for the endogenous gene the isolated 5'-flanking region of the villin gene responds positively to conditions known to stimulate terminal differentiation of these cultured epithelial intestinal cells. The reported results indicate that this genomic fragment contains sufficient regulatory elements to recapitulate the expression pattern of the villin promoter during intestinal differentiation.

Adenocarcinoma↗
PubMed1995

A regulatory element downstream of the rat SM22 alpha gene transcription start point enhances reporter gene expression in vascular smooth muscle cells.

SM22 alpha is a 22-kDa protein of unknown function, the mRNA of which is highly and specifically expressed in smooth muscle cells (SMC). The 5' untranslated leader sequence of the rat SM22 alpha gene was found to contain two introns of 3.6 and 2.9 kb. Two transcripts of SM22 alpha exist in all SMC types examined, and genomic mapping of the gene suggests these transcripts result from different 5' transcription start points, split by the 2.9-kb intron. A small intron (102 bp), which contains an E-box consensus sequence, was found within the coding region 178 bp from the ATG start codon. The 3.6-kb intron contains 82 bp which show 98% homology at the RNA level with the rat identifier sequence (ID). Transient reporter gene assays demonstrate that a 576-bp fragment, including the ID, contains a regulatory element which may contribute to the SMC-specific expression of SM22 alpha.

Animals↗
PubMed2006

Potential of the FES-hERL PET reporter gene system -- basic evaluation for gene therapy monitoring.

PURPOSE: In vivo reporter genes can be powerful tools in supporting and ensuring the success of gene therapy. A careful and rational design of a reporter system is essential to realize a noninvasive in vivo reporter gene imaging system applicable for humans. We designed a new in vivo reporter gene imaging system that uses F-18-labeled estradiol (FES) and human estrogen receptor ligand (hERL) binding domain, taking advantage that FES is a radiopharmaceutical already being used for human studies with access to a wide range of tissues, including the brain, and that hERL lacking DNA binding domain can no longer work as a transcription factor, and carried out basic studies to evaluate its potential for gene therapy monitoring. METHODS: We constructed a plasmid (pTIER) to coexpress a model therapeutic gene and the reporter gene hERL and transfected Cos7 cells and examined their uptake of [(3)H]estradiol and FES in culture media. The uptake of FES by mouse calf muscle electroporated with pTIER was also tested. RESULTS: The cells transfected with pTIER took up the radioligands efficiently and specifically in culture media. Also, the mouse calf muscle electroporated with pTIER accumulated a higher amount of FES than did the control. CONCLUSION: The data indicate that our new reporter gene system seems promising for in vivo imaging of gene expression and gene therapy monitoring.

Animals↗
PubMed2006

Investigating gene expression: in situ hybridization and reporter genes.

Coordinated cell type differentiation is essential for morphogenesis during Dictyostelium development. The specification of different cell types and the regulation of temporal and spatial patterns of expression of cell type-specific genes are important problems currently being addressed in many laboratories. Besides, determination of gene expression patterns provides significant information in the characterization of developmental mutants. Cell type-specific probes and well characterized promoters are available that allow the identification of most cell types during Dictyostelium development. Expression patterns can be studied by whole-mount in situ messenger RNA (mRNA) detection and by the use of reporter genes under the control of specific promoters. The most common in situ hybridization technique, based on nonradioactive ribo-probes that are hybridized to fixed whole-mounts prepared at different developmental stages, is described. Several reporter genes have been used to characterize gene expression patterns and to determine functional promoter elements. The lacZ gene, coding for the beta-galactosidase enzyme, is the reporter most frequently used in Dictyostelium because both temporal- and spatial-patterns of expression can be easily determined. Generally used beta-galactosidase detection methods are described.

Animals↗
PubMed2001

Identification of environmental stress-regulated genes in Helicobacter pylori by a lacZ reporter gene fusion system.

BACKGROUND: Helicobacter pylori persists in the human stomach for decades. This requires an efficient adaptation of H. pylori to the gastric niche and involves the regulation of bacterial genes in response to environmental stress. Efficient molecular tools to identify regulated H. pylori genes are scarce, therefore we developed a genomic lacZ reporter gene fusion system in H. pylori to screen for stress-regulated genes. MATERIALS AND METHODS: The integration vector pBW was constructed and used to generate random genomic lacZ fusions in H. pylori. Two-hundred-and-fifty H. pylori transformants were selected from this library, replica-plated and screened for differential lacZ expression after exposure to two environmental stress conditions: increased temperature (42 degrees C), and iron-limitation. RESULTS: From a library of H. pylori transformants with random genomic transcriptional lacZ fusions, two stress-regulated H. pylori loci were identified. The transcription of a gene of unknown function (designated hsp12) was increased by incubation at 42 degrees C. The transcription of a locus, consisting of the three fumarate reductase subunit genes (frdCAB) and the HP0190 gene from H. pylori strain 26695, was decreased under iron-limitation. CONCLUSIONS: This is the first time that a genomic transcriptional lacZ reporter gene H. pylori library has been used as a tool for the fast and efficient identification of environmental stress-regulated H. pylori genes.

Bacterial Proteins↗
PubMed1996

The 5' regulatory region from the Drosophila pseudoobscura hsp82 gene results in a high level of reporter gene expression in Lucilia cuprina embryos.

We have previously examined the efficiency of two Drosophila melanogaster promoters to enable reporter gene expression in embryos of the Australian sheep blowfly, Lucilia cuprina. Both the hsp70 heat-shock promoter and the actin5C promoter resulted in low levels of expression of a reporter gene in these embryos. In this study, the D. pseudoobscura hsp82 promoter (phsp82) was tested for its ability to direct the expression of the Escherichia coli chloramphenicol acetyltransferase-encoding gene (cat). We report that the level of CAT activity in L. cuprina embryos was comparable to that obtained with the same construct in D. melanogaster, indicating that phsp82 functions efficiently in this non-drosophilid insect. The results suggest that phsp82 may be utilised in other non-drosophilid insects in which poor expression levels are obtained from constructs containing the hsp70 or actin5C promoters.

Animals↗
PubMed2005

Cardiac reporter gene imaging using the human sodium/iodide symporter gene.

OBJECTIVE: Imaging of reporter gene expression holds promise for noninvasive monitoring of cardiovascular molecular therapy. We investigated the feasibility of myocardial gene expression imaging in living rats using the human sodium/iodide symporter gene (hNIS) and widely available scintigraphic techniques. METHODS: We injected adenovirus expressing hNIS under control of cytomegalovirus promoter (Ad(hNIS)) directly into left ventricular myocardium of Wistar rats. For detection of reporter gene expression, dynamic gamma-camera imaging was performed following intravenous injection of (123)Iodide or (99m)Technetium. RESULTS: For both radiotracers, focal cardiac accumulation was identified as early as 10 min, and remained detectable until 2 hrs after injection, while it was not present in animals injected with LacZ control virus. Intensity of tracer accumulation gradually decreased when decreasing titers of Ad(hNIS) were applied. Treatment with sodium perchlorate (a blocker of hNIS) abolished cardiac tracer uptake after Ad(hNIS)-infection. Serial imaging after cardiac gene transfer demonstrated a peak of tracer signal between days 1 and 3, and a subsequent decrease until day 12. Postmortem analysis of hearts yielded significant correlation between in vivo radiotracer accumulation and ex vivo gamma-counting. Autoradiography demonstrated specific regional radioactivity in Ad(hNIS)-infected myocardial areas. CONCLUSIONS: hNIS offers a practical and reliable approach for myocardial gene expression imaging. Using suitable vectors, hNIS may be coexpressed with therapeutic genes or stably expressed in stem cells for future monitoring of cardiovascular molecular therapy.

Animals↗
PubMed1998

Deletion of the Saccharomyces cerevisiae TRR1 gene encoding thioredoxin reductase inhibits p53-dependent reporter gene expression.

The prevalence of p53 gene mutations in many human tumors implies that p53 protein plays an important role in preventing cancers. Central among the activities ascribed to p53 is its ability to stimulate transcription of other genes that inhibit cells from entering S phase with damaged DNA. Human p53 can be studied in yeast where genetic tools can be used to identify proteins that affect its ability to stimulate transcription. Although p53 strongly stimulated reporter gene expression in wild type yeast, it only weakly stimulated reporter gene expression in Deltatrr1 yeast that lacked the gene encoding thioredoxin reductase. Furthermore, ectoptic expression of TRR1 in Deltatrr1 yeast restored p53-dependent reporter gene activity to high levels. Immunoblot assays established that the Deltatrr1 mutation affected the activity and not the level of p53 protein. The results suggest that p53 can form disulfides and that these disulfides must be reduced in order for the protein to function as a transcription factor.

DNA-Binding Proteins↗
PubMed1997

Sequences in the proximal 5' flanking region of the rat neuron-specific enolase (NSE) gene are sufficient for cell type-specific reporter gene expression.

We investigated the regulation of the rat neuron-specific enolase gene using a transient transfection approach. Recent transgenic mouse studies have shown that a 1.8-kb segment of the rat NSE gene 5' flanking region, including the first (noncoding) exon but not the first intron, is able to drive expression of a reporter gene in parallel with endogenous NSE. These data suggest that cis-acting elements responsible for the spatial and temporal pattern of NSE gene expression are located within the proximal 1.8 kb of the 5' flanking sequence. To further investigate this region, we joined the 1.8-kb regulatory cassette to the cat reporter gene and generated a number of constructs in which the flanking sequence was progressively deleted from the 5' end. These constructs were tested by transient transfection into neuronal and nonneuronal cells, followed by an assay for CAT activity. We found that as little as 255 bp of 5' flanking sequence was able to confer cell type-specificity on the reporter gene. Further truncation to 120 bp of 5' sequence resulted in a sharp downregulation of reporter activity in PC12 cells but a significant rise in both Neuro-2A neuroblastoma cells and nonneuronal Ltk- cells, indicating that cis-acting elements controlling the regulation of NSE in Ltk-, Neuro-2A, and PC12 cells may lie within the 135 bp region covered by this deletion. This region contains an AP-2 site and an element similar in sequence and position to a motif identified in the proximal promoter region of the neuron-specific peripherin gene. Reduction to 95 bp of 5' sequence resulted in a slight downregulation of CAT activity in all cell lines tested, and further truncation to 65 bp of 5' sequence caused a universal reduction to background levels of CAT activity, concomitant with the disruption of the basal NSE promoter. Our results show that the 5' flanking region of the NSE gene is capable of conferring cell type-specificity on a heterologous gene in transfected cells and that elements responsible for this are located within the proximal 255 bp.

Animals↗
PubMed2002

A 3-kilobase region derived from the rat cathepsin L gene directs in vivo expression of a reporter gene in sertoli cells in a manner comparable to that of the endogenous gene.

During mammalian spermatogenesis, the transcription of several genes in Sertoli cells is turned on and off as the adjacent male germ cells progress through the stages of the cycle of the seminiferous epithelium. A requirement for defining how germ cells regulate this process is the identification of a promoter that confers, in vivo, accurate, stage-specific gene expression in Sertoli cells. To date, such a promoter has not been identified. Using transgenic mice, we show that the 3-kilobase genomic fragment immediately upstream of the rat cathepsin L translation start site directs expression of the reporter gene, beta-galactosidase, only in Sertoli cells. The expression pattern of the reporter gene recapitulated that of the endogenous gene in Sertoli cells as 75% of the seminiferous tubules that contained X-gal positive Sertoli cells were at stages VI-VIII and beta-galactosidase enzymatic activity was 4-fold higher in mature testes compared with immature testes. This is, to our knowledge, the first identification of a promoter region that contains all of the regulatory elements required for accurate, stage-specific gene expression in Sertoli cells.

Animals↗
PubMed1998

The use of a thermostable beta-glucanase gene from Clostridium thermocellum as a reporter gene in plants.

In order to take advantage of the high thermostability of its product, beta-1,3;1,4-glucanase (lichenase), we used a modified version of the licB gene from Clostridium thermocellum as a reporter gene for the analysis of gene expression in transformed plants. The coding region of the licB gene was truncated at both ends. The truncated enzyme retained its activity and thermostability. The modified gene (m-licB), with and without a plant leader peptide-encoding sequence, was expressed in tobacco plants under control of either the Agrobacterium octopine TR-DNA 2' gene promoter or the promoter of the gene for the small subunit of ribulose-1,5-bisphosphate carboxylase. Expression of licB can be measured quantitatively and accurately, the assay is sensitive and simple enough to be used for analysis of various gene fusion systems or for screening of transformants. The enzyme is very stable and remains active in tissue extracts even after storage for 1 year and survives many thawing-freezing cycles. The lichenase-encoding gene was expressed at high levels in transformed tobacco plants without any apparent detrimental effects on vegetative growth or flowering.

Clostridium↗
PubMed1998

The treA gene of Bacillus subtilis is a suitable reporter gene for the archaeon Methanococcus voltae.

The similarity of the transcriptional apparatus of Archaea with that of Eucarya makes studies of their transcriptional regulation especially interesting. Such investigations are greatly facilitated by reporter genes. The concomitant analysis of several promoters for investigations of regulatory patterns requires different reporter genes. The archaeon Methanococcus voltae is a moderately halophilic mesophile. The treA gene from Bacillus subtilis appeared to be a good candidate for a reporter, since its product trehalase is salt-resistant. We show that it is indeed expressed under the control of a M. voltae promoter and that the enzyme is easily testable in cell lysates.

Bacillus subtilis↗
PubMed2002

The effect of DNA-alkylating agents on gene expression from two integrated reporter genes in a mouse mammary tumor line.

A model system was developed to investigate the effects of DNA alkylating agents on cellular gene expression. The cytomegalovirus immediate-early promoter (CMV) and the mouse mammary tumor virus promoter (MMTV) were coupled separately to the luciferase reporter gene and stably expressed in cultured cells. The change in luciferase activity was used as a measure of gene expression inhibition. Seven well-characterized DNA alkylating agents of varied DNA adduct-forming ability were evaluated in this system. The major groove binders/intercalators (that form guanine adducts) increased CMV-luciferase activity above background, while minor groove binders (that form adenine adducts) all decreased it. The MMTV-luciferase activity was remarkably different to the CMV-luciferase activity and was inhibited to the greatest extent by the minor groove alkylators. One of these, a polybenzamide with spatially separated alkylating groups, inhibited gene expression to a greater extent than inhibition of general DNA or RNA synthesis.

Alkylating Agents↗
PubMed1992

Chorion gene cis-regulatory DNA restricts tissue specificity of reporter gene expression in transformed Drosophila.

P element mediated germ-line transformation was used to study the developmental specificity of Drosophila chorion gene regulatory sequences directing expression of the bacterial reporter genes for chloramphenicol acetyltransferase (CAT) and beta-galactosidase (lacZ). DNA fragments containing 5' flanking plus the entire 5' untranslated and the beginning of the coding region of either the s36 or the s15 chorion gene are able to confer on the reporter genes normal tissue as well as temporal specificity of expression, exclusively in the ovary of transformed female flies. However, if 5' untranslated and coding regions are omitted, normal ovarian expression is maintained but tissue specificity is relaxed: expression of the reporter gene is detected both in the ovary and in specific non-ovarian tissues of transformed females and males. The evidence suggests that the missing 5' untranslated and coding sequences may include negative elements that normally suppress expression in non-ovarian tissues, and that these putative elements are distinct from those that prevent premature expression in the ovarian follicles. The exact location of ectopic lacZ expression within the internal male genitalia depends on the constellation of 5' flanking chorion regulatory sequences included in the P element constructs. Ectopic expression of the CAT gene in the male genitalia under s15 promoter control can be abolished by mutating the hexamer TCACGT, a sequence previously shown to be essential for the normal expression of this chorion gene in the ovary.

Animals↗
PubMed2005

Molecular analysis of Arabidopsis endosperm and embryo promoter trap lines: reporter-gene expression can result from T-DNA insertions in antisense orientation, in introns and in intergenic regions, in addition to sense insertion at the 5' end of genes.

Random insertions of promoterless reporter genes in genomes are a common tool for identifying marker lines with tissue-specific expression patterns. Such lines are assumed to reflect the activity of endogenous promoters and should facilitate the cloning of genes expressed in the corresponding tissues. To identify genes active in seed organs, plant DNA flanking T-DNA insertions (T-DNAs) have been cloned in 16 Arabidopsis thaliana GUS-reporter lines. T-DNAs were found in proximal promoter regions, 5' UTR or intron with GUS in the same (sense) orientation as the tagged gene, but contrary to expectations also in inverted orientation in the 5' end of genes or in intergenic regions. RT-PCR, northern analysis, and data on expression patterns of tagged genes, compared with the expression pattern of the reporter lines, suggest that the expression pattern of a reporter gene will reflect the pattern of a tagged gene when inserted in sense orientation in the 5' UTR or intron. When inserted in the promoter region, the reporter-gene expression patterns may be restricted compared with the endogenous gene. Among the trapped genes, the previously described nitrate transporter gene AtNRT1.1, the cyclophilin gene ROC3, and the histone deacetylase gene AtHD2C were found. Reporter-gene expression when positioned in antisense orientation, for example, in the SLEEPY1 gene, is indicative of antisense expression of the tagged gene. For T-DNAs found in intergenic regions, it is suggested that the reporter gene is transcribed from cryptic promoters or promoters of as yet unannotated genes.

Arabidopsis↗
PubMed2003

[Construction of pseudorabies virus SH strain with gE-gI gene partial deletion mutant including GFP reporter gene].

On the basis of cloning and indentifying gE-gI gene of pseudurabie virus SH strain, the transfer plasmid vector was constucted in order to get the gE-gI gene partial deletion mutant. At first, gE gene and gI gene were cloned into pUC18, constructed the pgEI vector. Then, the 5' terminal sequence of gE gene was deleted 363bp using the restrict endonuclease in gE gene. The GFP expressing cassette was inserted into the deleting site. The recombinant plasmid pgEI including GFP reporter gene deleted part of gE-gI gene was constructed. BHK-21 cell which was infected with PRV-SH for 1-2h were tansfected with the complex of pgEI-GFP and DOTAPA deletion mutant was selected and purified many times in BHK-21 cell through GFP. Inoculation of mice with 2.0X107 PFU of the recombinant virus revealed that mice were partly protected against challenge with PRV-SH containing 2MLD.

Animals↗