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PubMed1997

Naturally occurring mutations in the human 5-lipoxygenase gene promoter that modify transcription factor binding and reporter gene transcription.

Five lipoxygenase (5-LO) is the first committed enzyme in the metabolic pathway leading to the synthesis of the leukotrienes. We examined genomic DNA isolated from 25 normal subjects and 31 patients with asthma (6 of whom had aspirin-sensitive asthma) for mutations in the known transcription factor binding regions and the protein encoding region of the 5-LO gene. A family of mutations in the G + C-rich transcription factor binding region was identified consisting of the deletion of one, deletion of two, or addition of one zinc finger (Sp1/Egr-1) binding sites in the region 176 to 147 bp upstream from the ATG translation start site where there are normally 5 Sp1 binding motifs in tandem. Reporter gene activity directed by any of the mutant forms of the transcription factor binding region was significantly (P < 0.05) less effective than the activity driven by the wild type transcription factor binding region. Electrophoretic mobility shift assays (EMSAs) demonstrated the capacity of wild type and mutant transcription factor binding regions to bind nuclear extracts from human umbilical vein endothelial cells (HUVECs). These data are consistent with a family of mutations in the 5-LO gene that can modify reporter gene transcription possibly through differences in Sp1 and Egr-1 transactivation.

Alleles↗

PubMed1998

Downstream elements from the pea albumin 1 gene confer sulfur responsiveness on a reporter gene.

The levels of mRNAs for some of the sulfur-rich proteins in seeds are regulated by the level of sulfur supplied to the plants. In peas, there is a mechanism that lowers the level of mRNA for legumin and pea albumin 1 (PA1) when plants are grown under sulfur-deficient conditions. This mechanism acts after transcription initiation. In this study, a gene encoding PA1 was expressed in leaves of transgenic tobacco. Expression of the gene was controlled by the level of sulfur supplied to the plants, mimicking the behaviour of the intact gene in peas. A gene encoding a different high-sulfur protein, ovalbumin, was unresponsive to sulfur status and was used as a reporter gene to test defined regions of the PA1 gene for sulfur responsiveness. These constructs, together with a set of PA1 gene deletions, were tested in transgenic tobacco and yielded the following observations: the PA1 gene was sensitive to sulfur status in the leaf as well as the seed; intron processing of the PA1 transcript was not required for sensitivity to sulfur stress; both the coding region and the 3' flanking regions of the PA1 gene contained sequences which conferred sensitivity to sulfur stress; the sulfur-responsive sequence in the 3' region was contained within a 134-nucleotide segment downstream of the end of the coding sequence. We conclude that there are at least two downstream elements which confer sensitivity to sulfur supply.

Albumins↗

PubMed2000

Infection by bacterial pathogens expressing type III secretion decreases luciferase activity: ramifications for reporter gene studies.

Pathogenic microbes influence gene regulation in eukaryotic hosts. Reporter gene studies can define the roles of promoter regulatory sequences. The effect of pathogenic bacteria on reporter genes has not been examined. The aim of this study was to identify which reporter genes are reliable in studies concerning host gene regulation by bacterial pathogens expressing type III secretory systems. Human intestinal epithelial cells, T84, Caco-2 and HT-29, were transfected with plasmids containing luciferase (luc), chloramphenicol acetyltransferase (CAT) or beta-galactosidase (beta-gal) as reporter genes driven by the inducible interleukin-8 (IL-8) or constitutively active simian virus 40 (SV40) promoter. Cells were infected with enteropathogenic E. coli or Salmonella typhimurium, and the reporter activity was assessed. Luc activity significantly decreased following infection, regardless of the promoter. The activity of recombinant luc was nearly ablated by incubation with either EPEC or Salmonella in a cell-free system. Activity was partially preserved by protease inhibitors, and immunoblot analysis showed a decreased amount and molecular weight of recombinant luc, suggesting protein degradation. Neither beta-gal nor CAT activity was altered by infection. Disruption of type III secretion prevented the loss of luc activity. We conclude that CAT or beta-gal, but not luc, can be used as reliable reporter genes to assess the impact of pathogenic microbes, especially those expressing type III secretion on host cell gene regulation.

Cell Line↗

PubMed2001

[In vitro and in vivo experiment of transduction of reporter gene into tumors by E5 gene delivery system].

OBJECTIVE: To investigate the spectrum of tumors into which the E5 gene delivery system can transduce reporter gene so as to establish a platform technology that one gene delivery system can be used to treat multiple types of tumors. METHODS: The E5 delecvery system was used to transduce reporter genes into various types of IGF I R positive tumor cell lines in vitro and into different IGF I R positive tumors transplanted subcutaneously in nude mice in vivo. The efficiency of trasduction was examined. RESULTS: The E5 delivery system transferred reporter genes into five types of tumor cells that over-express IGFIR in vitro and transferred reporter genes into different tumors transplanted subcutaneously in nude mice that over-express IGFIR in vivo too. The transduction rate was positively correlated with the expression rate of IGFIR. However, the E5 delivery system failed to transfer reporter genes into tumor cedll lines in vitro and the tumors transplanted subcutaneously in nude mice in vivo, both of which were without IGF I R expression. CONCLUSION: E5 delivery system has rather remarkable target-ability and extensive usefulness. It has the potentiality in gene therapy of cancer.

Animals↗

PubMed2001

Optical imaging of Renilla luciferase reporter gene expression in living mice.

Imaging reporter gene expression in living subjects is a rapidly evolving area of molecular imaging research. Studies have validated the use of reporter genes with positron emission tomography (PET), single photon emission computed tomography (SPECT), MRI, fluorescence with wild-type and mutants of green fluorescent protein, as well as bioluminescence using Firefly luciferase enzyme/protein (FL). In the current study, we validate for the first time the ability to image bioluminescence from Renilla luciferase enzyme/protein (RL) by injecting the substrate coelenterazine in living mice. A highly sensitive cooled charge-coupled device camera provides images within a few minutes of photon counting. Cells, transiently expressing the Rluc were imaged while located in the peritoneum, s.c. layer, as well as in the liver and lungs of living mice tail-vein injected with coelenterazine. Furthermore, d-luciferin (a substrate for FL) does not serve as a substrate for RL, and coelenterazine does not serve as a substrate for FL either in cell culture or in living mice. We also show that both Rluc and Fluc expression can be imaged in the same living mouse and that the kinetics of light production are distinct. The approaches validated will have direct applications to various studies where two molecular events need to be tracked, including cell trafficking of two cell populations, two gene therapy vectors, and indirect monitoring of two endogenous genes through the use of two reporter genes.

Animals↗

PubMed2002

Positron emission tomography imaging of cardiac reporter gene expression in living rats.

BACKGROUND: Imaging reporter gene expression is useful for noninvasive monitoring of gene therapy. In this study, we imaged cardiac reporter gene expression in living rats using micro positron emission tomography (microPET). METHODS AND RESULTS: Rats (n=10) underwent intramyocardial injection with 1x10(9) pfu of adenovirus carrying cytomegalovirus promoter-driving herpes simplex virus type 1 mutant thymidine kinase (Ad-CMV-HSV1-sr39tk) as PET reporter gene. Control rats (n=4) received 1x10(9) pfu of adenovirus carrying cytomegalovirus promoter-driving firefly luciferase (Ad-CMV-Fluc). On days 2 to 4, microPET images were obtained after a tail vein injection of nitrogen-13 ammonia ([13N]-NH3) as myocardial perfusion tracer, followed by 9-(4-[18F]-fluoro-3 hydroxymethylbutyl) guanine ([18F]-FHBG) to assess HSV1-sr39tk expression. After imaging, hearts were removed for ex vivo [18F] gamma counting and thymidine kinase enzyme assay. Results show homogenous myocardial distribution of [13N]-NH3 on all microPET images. Rats injected with Ad-CMV-HSV1-sr39tk have significant [18F]-FHBG uptake in the anterolateral wall compared with background signal in controls. Gamma counting shows 20.0+/-4.4-fold increase of radioactivity, whereas enzyme assay shows 22.1+/-6.1-fold increase of thymidine kinase activity in Ad-CMV-HSV1-sr39tk injected rats (P<0.05). CONCLUSIONS: Successful imaging of cardiac HSV1-sr39tk expression was performed in living rats with microPET. The presence of [18F]-FHBG uptake is confirmed by gamma counting and the presence of HSV1-sr39TK protein by thymidine kinase enzyme assay. Cardiac reporter gene imaging by PET may eventually be applied toward human gene therapy studies.

Adenoviridae↗

PubMed2002

Development of a GFP reporter gene for Chlamydomonas reinhardtii chloroplast.

Reporter genes have been successfully used in chloroplasts of higher plants, and high levels of recombinant protein expression have been reported. Reporter genes have also been used in the chloroplast of Chlamydomonas reinhardtii, but in most cases the amounts of protein produced appeared to be very low. We hypothesized that the inability to achieve high levels of recombinant protein expression in the C. reinhardtii chloroplast was due to the codon bias seen in the C. reinhardtii chloroplast genome. To test this hypothesis, we synthesized a gene encoding green fluorescent protein (GFP) de novo, optimizing its codon usage to reflect that of major C. reinhardtii chloroplast-encoded proteins. We monitored the accumulation of GFP in C. reinhardtii chloroplasts transformed with the codon-optimized GFP cassette (GFPct), under the control of the C. reinhardtii rbcL 5'- and 3'-UTRs. We compared this expression with the accumulation of GFP in C. reinhardtii transformed with a non-optimized GFP cassette (GFPncb), also under the control of the rbcL 5'- and 3'-UTRs. We demonstrate that C. reinhardtii chloroplasts transformed with the GFPct cassette accumulate approximately 80-fold more GFP than GFPncb-transformed strains. We further demonstrate that expression from the GFPct cassette, under control of the rbcL 5'- and 3'-UTRs, is sufficiently robust to report differences in protein synthesis based on subtle changes in environmental conditions, showing the utility of the GFPct gene as a reporter of C. reinhardtii chloroplast gene expression.

Amino Acid Sequence↗

PubMed2000

Cambial-region-specific expression of the Agrobacterium iaa genes in transgenic aspen visualized by a linked uidA reporter gene.

The level of indole-3-acetic acid (IAA) was locally modified in cambial tissues of transgenic aspen (Populus tremula L. x Populus tremuloides Michx.). We also demonstrate the use of a linked reporter gene to visualize the expression of the iaa genes. The rate-limiting bacterial IAA-biosynthetic gene iaaM and the reporter gene for beta-glucuronidase (GUS), uidA, were each fused to the cambial-region-specific Agrobacterium rhizogenes rolC promoter and linked on the same T-DNA. In situ hybridization of the iaaM gene confirmed that histochemical analysis of GUS activity could be used to predict iaaM gene expression. Moreover, quantitative fluorometric analysis of GUS activity allowed estimation of the level of de novo production of IAA in transgenic lines carrying a single-copy insert of the iaaM, uidA T-DNA. Microscale analysis of the IAA concentration across the cambial region tissues showed an increase in IAA concentration of about 35% to 40% in the two transgenic lines, but no changes in the radial distribution pattern of IAA compared with wild-type plants. This increase did not result in any changes in the developmental pattern of cambial derivatives or the cambial growth rate, which emphasizes the importance of the radial distribution pattern of IAA in controlling the development of secondary xylem, and suggests that a moderate increase in IAA concentration does not necessarily stimulate growth.

Blotting, Northern↗

PubMed2000

Identification of a short cis-acting element in the human vasopressin type 2 receptor gene which confers high-level expression of a reporter gene specifically in collecting duct cells.

In the kidney, water reabsorption is mainly regulated by the binding of arginine vasopressin to vasopressin type 2 (V2) receptors. These receptors are expressed selectively in principal cells of the collecting ducts. To identify molecular mechanisms responsible for the cell-specific expression of the V2 receptor, we have analyzed the proximal promoter of the corresponding gene. We report the identification of a 33-bp enhancer [collecting duct tissue-specific element 1 (CSE1)] that induced high levels of expression of the luciferase reporter gene in three collecting duct cell lines, but not in other renal cell lines. In gel shift assays, CSE1 bound a DNA-binding protein expressed selectively in collecting duct cell lines, and a 7-bp mutation, which abolished the activity of CSE1 in transient transfection experiments, also abolished the binding of this protein. Furthermore, decoy experiments performed using CSE1 showed that this sequence was involved not only in the expression of a construct containing 4.2 kb of the V2 receptor proximal promoter, but also in the expression of the endogenous V2 receptor gene. CSE1 appears to act mostly by counteracting the inhibitory effects of a strong ubiquitous repressor element that we called CIE1. Collectively, these results identify the first functional collecting duct-specific cis-acting element.

3T3 Cells↗

PubMed1996

Assessing the estrogenic and dioxin-like activities of chemicals and complex mixtures using in vitro recombinant receptor-reporter gene assays.

In vitro recombinant receptor-reporter gene assays have been used to assess and rank the potency of chemicals and complex mixtures suspected of possessing estrogen and (or) aryl hydrocarbon receptor (AhR) mediated activity. The environmental estrogen (E2) bioassay consists of a Gal4-human estrogen receptor chimeric construct (Gal4-HEGO) and a Gal4-regulated luciferase reporter gene (17m5-G-Luc) that have been stably integrated into HeLa cells. The assay exhibits 10-fold induction in luciferase reporter gene activity following treatment with 1 nM 17 beta-estradiol and has a detection limit of approximately 5 pg of 17 beta-estradiol/mL. The AhR bioassay uses Hepa 1c1c7 wild-type cells transiently transfected with a dioxin response element regulated luciferase reporter gene. These assays were used to assess the estrogen and dioxin-like activities of naringenin, atrazine, and simazine and complex mixtures such as pulp and paper mill black liquor and urban air particulates. The activities of these chemicals and complex mixtures are confirmed using the pure antiestrogen ICI 164,384 and in in vitro gel retardation assays. Results of this study demonstrate the utility of in vitro recombinant receptor-reporter gene assays in identifying and assessing the estrogenic and dioxin-like activities of chemicals and complex mixtures.

Air Pollutants↗

PubMed2000

Seeing is believing: non-invasive, quantitative and repetitive imaging of reporter gene expression in living animals, using positron emission tomography.

The ability to monitor reporter gene expression in living animals and in patients will permit longitudinal examinations both of somatically transferred DNA in experimental animals and patients and of transgenic constructs expressed in experimental animals. If investigators can non-invasively monitor the organ and tissue specificity, the magnitude and the duration of gene expression from somatically transferred DNA and from transgenes, conceptually new experimental paradigms will be possible. If clinicians can non-invasively monitor the location, extent and duration of somatically transferred genes, they will be better able to determine the correlations between expression of therapeutic genes and clinical outcomes. We have developed two reporter gene systems for in vivo reporter gene imaging in which the protein products of the reporter genes sequester positron-emitting reporter probes. The "PET reporter gene" dependent sequestration of the "PET reporter probes" is subsequently measured in living animals by Positron Emission Tomography (PET). We describe here the principles of PET reporter gene/PET reporter probe in vivo imaging, the development of two imaging systems, and the validation of their ability to non-invasively, quantitatively and repetitively image reporter gene expression in murine viral gene transfer and transgenic models.

Animals↗

PubMed1999

Reporter gene technology: the future looks bright.

Reporter gene technology is widely used to monitor the cellular events associated with signal transduction and gene expression. Based upon the splicing of transcriptional control elements to a variety of reporter genes (with easily measurable phenotypes), it "reports" the effects of a cascade of signalling events on gene expression inside cells. The principal advantage of these assays is their high sensitivity, reliability, convenience, and adaptability to large-scale measurements. This review summarises the current status of reporter gene technology including its role in monitoring gene transfer and expression and its development as a biological screen. With the advances in this technology and in detection methods, it is likely that luciferase and green fluorescent protein will become increasingly popular for the non-invasive monitoring of gene expression in living tissues and cells. Such techniques will be important in defining the molecular events associated with gene transcription, which has implications for our understanding of the molecular basis of disease and will influence our approach to gene therapy and drug development.

Drug Design↗

PubMed2004

Novel bidirectional vector strategy for amplification of therapeutic and reporter gene expression.

Molecular imaging methods have previously been employed to image tissue-specific reporter gene expression by a two-step transcriptional amplification (TSTA) strategy. We have now developed a new bidirectional vector system, based on the TSTA strategy, that can simultaneously amplify expression for both a target gene and a reporter gene, using a relatively weak promoter. We used the synthetic Renilla luciferase (hrl) and firefly luciferase (fl) reporter genes to validate the system in cell cultures and in living mice. When mammalian cells were transiently cotransfected with the GAL4-responsive bidirectional reporter vector and various doses of the activator plasmid encoding the GAL4-VP16 fusion protein, pSV40-GAL4-VP16, a high correlation (r(2) = 0.95) was observed between the expression levels of both reporter genes. Good correlations (r(2) = 0.82 and 0.66, respectively) were also observed in vivo when the transiently transfected cells were implanted subcutaneously in mice or when the two plasmids were delivered by hydrodynamic injection and imaged. This work establishes a novel bidirectional vector approach utilizing the TSTA strategy for both target and reporter gene amplification. This validated approach should prove useful for the development of novel gene therapy vectors, as well as for transgenic models, allowing noninvasive imaging for indirect monitoring and amplification of target gene expression.

Animals↗

PubMed2000

Development of genetic tools for Lactobacillus sakei: disruption of the beta-galactosidase gene and use of lacZ as a reporter gene To study regulation of the putative copper ATPase, AtkB.

Downstream from the ptsHI operon of Lactobacillus sakei, the genes atkY and atkB, organized in an operon, were observed. The two putative proteins, AtkB and AtkY, show sequence similarity to the Enterococcus hirae copper P-type ATPase, responsible for copper efflux, and its negative regulator. Characterization of AtkB as a copper P-type ATPase could not be demonstrated since an atkB mutant did not show any phenotype. Thus, another strategy was followed in order to investigate the transcriptional regulation of the atkYB locus, leading to the development of new genetic tools for L. sakei. A plasmid was constructed, the use of which allowed gene replacement at the lacLM locus in L. sakei by two successive crossovers. A strain deleted of the lacLM operon encoding the beta-galactosidase of L. sakei was constructed by this method, and the Escherichia coli lacZ gene could then be used as a reporter gene to investigate the regulation of atkYB. Results show that the atkYB operon is induced by small concentrations of CuSO(4) (30 to 40 microM) but not when CuSO(4) is omitted or added at higher concentrations.

Adenosine Triphosphatases↗

PubMed2003

Optical bioluminescence and positron emission tomography imaging of a novel fusion reporter gene in tumor xenografts of living mice.

Noninvasive imaging of reporter gene expression using various imaging modalitiesis playing an increasingly important role in defining molecular events in the field of cancer biology, cell biology, and gene therapy. In this study, a novel reporter vector was constructed encoding a fusion protein comprised of a mutant herpes simplex virus type 1 thymidine kinase (HSV1-sr39tk) (tk), a positron emission tomography (PET) reporter gene, and renilla luciferase (rl), a bioluminescence optical reporter gene joined by a 20 amino acid long spacer sequence. We validated the activity of the two enzymes encoded by the fusion protein (tk(20)rl) in cell culture. Then, tumors stably expressing the tk(20)rl fusion gene were imaged both by microPET and optically using a cooled charge coupled device camera in xenograft-bearing living mice. Using a single fusion reporter (PET/optical) gene should accelerate the validation of reporter gene approaches developed in cell culture for translation into preclinical and clinical models.

Amino Acid Sequence↗

PubMed2002

Reporter gene transfer induces apoptosis in primary cortical neurons.

Modern cell biologists typically use reporter genes either alone or co-expressed with a protein of interest to facilitate the localization or quantification of protein expression. Our work demonstrates that reporter genes should be used cautiously, as several common reporter gene products are toxic to primary cortical neuronal cultures. We used the herpes simplex virus-based viral amplicon vector to transduce cortical neurons with three different reporter genes and assessed whether any reporter gene products were toxic over time, by monitoring neurite disintegration and apoptosis. Toxicity varied as a function of the reporter gene, the gene product localization, and the level of reporter gene expression. Transduction of enhanced green fluorescent protein or nuclear-localized beta-galactosidase was more toxic than non-nuclear localized beta-galactosidase. This work underscores the need for careful design of gene expression constructs. Moreover, in studies where cell injury or toxicity is being evaluated, their use should be carefully considered.

Animals↗

PubMed2002

Reporter gene regulation in Saccharomyces cerevisiae by the human p53 tumor suppressor protein.

This study evaluated the transcriptional regulation of four reporter genes in Saccharomyces cerevisiae by the human tumor suppressor protein p53. The S. cerevisiae ADE2, HIS3 and URA3 genes were used with nutritional selections and the E. coli LacZ gene was used to quantitate reporter gene activation. DNA elements containing binding sites for p53 were introduced upstream of several 5' truncated yeast promoters and used to express reporter genes. Human p53 cDNA was expressed at different levels by utilizing three different yeast promoters. All reporter genes were activated by p53, and in the case of nutritional selections, basal reporter gene expression could be detected in the absence of p53. A gap repair assay was evaluated and optimized for the purpose of determining whether p53 encoded in various cDNA sources was functional in transcriptional transactivation. The basal levels of reporter gene transcription in the absence of p53 could be decreased by integration of the reporter gene in the chromosome. For several expression systems, p53 appears to be limiting since higher levels of reporter gene expression were observed when the p53 cDNA was expressed from more efficient promoters. The gap repair assay can be used to determine the genotype (homozygous wild type, homozygous mutant or heterozygous) for cDNA generated from human cell lines or tissue samples. This assay can also be used to evaluate mutation rates associated with various conditions for in vitro PCR amplification of DNA.

Cell Line↗

PubMed2006

Study of transcriptional regulation using a reporter gene assay.

Study of gene expression can be facilitated by using a reporter gene assay. Instead of directly measuring the level of target gene mRNA, one can clone the promoter region of the gene of interest in front of a reporter gene and measure the reporter gene expression as a reflection of the expression of the gene of interest. We describe a simple lacZ-fusion system to measure the activity of the reporter gene product beta-galactosidase. Different strategies of making the fusion construct and their applications are also discussed. This method is particularly useful to dissect the promoter region of the gene of interest and is also used in other experimental protocols such as the yeast two-hybrid analysis.

Artificial Gene Fusion↗