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Results for “Luciferases”

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PubMed1996

Sequence and biochemical similarities between the luciferases of the glow-worm Lampyris noctiluca and the firefly Photinus pyralis.

A full-length clone encoding Lampyris noctiluca (British glow-worm) luciferase was isolated from a complementary DNA (cDNA) expression library constructed with MRNA extracted from light organs. The luciferase was a 547-residue protein, as deduced from the nucleotide sequence. The protein was closely related to those of other lampyrid beetles, the similarity to Photinus pyralis luciferase being 84% and to Luciola 67%. In contrast, Lampyris luciferase had less sequence similarity to the luciferases of the click beetle Pyrophorus, at 48%. Engineering Lampyris luciferase in vitro showed that the C-terminal peptide containing 12 amino acids in Photinus and 9 amino acids in Lampyris was essential for bioluminescence. The pH optimum and the Km values for ATP and luciferin were similar for both Photinus and Lampyris luciferases, although the light emitted by the latter shifted towards the blue and was less stable at 37 degrees C. It was concluded that the molecular and biochemical properties were not sufficient to explain the glowing or flashing of the two beetles Lampyris and Photinus.

Adenosine Triphosphate↗

PubMed2004

Kinetics of regulated protein-protein interactions revealed with firefly luciferase complementation imaging in cells and living animals.

Signaling pathways regulating proliferation, differentiation, and apoptosis are commonly mediated through protein-protein interactions as well as reversible phosphorylation of proteins. To facilitate the study of regulated protein-protein interactions in cells and living animals, we optimized firefly luciferase protein fragment complementation by screening incremental truncation libraries of N- and C-terminal fragments of luciferase. Fused to the rapamycin-binding domain (FRB) of the kinase mammalian target of rapamycin and FK506-binding protein 12 (FKBP), respectively, the optimized FRB-N-terminal luciferase fragment (NLuc)/C-terminal luciferase fragment (CLuc)-FKBP luciferase complementation imaging (LCI) pair reconstituted luciferase activity in cells upon single-site binding of rapamycin in an FK506-competitive manner. LCI was used in three independent applications. In mice bearing implants of cells expressing the FRB-NLuc/CLuc-FKBP LCI pair, dose- and time-dependent luciferase activity allowed target-specific pharmacodynamic analysis of rapamycin-induced protein-protein interactions in vivo. In cells expressing a Cdc25C-NLuc/CLuc-14-3-3epsilon LCI pair, drug-mediated disruption of cell cycle regulated protein-protein interactions was demonstrated with the protein kinase inhibitor UCN-01 in a phosphoserine-dependent manner. When applied to IFN-gamma-dependent activation of Janus kinase/signal transducer and activator of transcription 1 (STAT1), LCI revealed, in the absence of ligand-induced phosphorylation, STAT1 proteins existing in live cells as preformed dimers. Thus, optimized LCI provides a platform for near real-time detection and characterization of regulated and small molecule-induced protein-protein interactions in intact cells and living animals and should enable a wide range of novel applications in drug discovery, chemical genetics, and proteomics research.

14-3-3 Proteins↗

PubMed1994

Transient transfection of the enteric parasite Entamoeba histolytica and expression of firefly luciferase.

Development of DNA-mediated transfection in Entamoeba histolytica will facilitate basic research toward the control of this protozoan parasite. A transient transfection system was established by using the firefly luciferase gene ligated to the 5' and 3' flanking regions of the amebic hgl1 gene. The optimal construct tested encoded an hgl1-luciferase fusion protein and contained 1 kb of 5' flanking sequence with 16 bases of coding sequence from the hgl1 gene ligated in-frame to the luciferase start codon and 2.3 kb of 3' flanking sequence from hgl1 ligated 3' to the luciferase stop codon. Optimal electroporation conditions in strain HM-1:IMSS trophozoites when using this construct were 500 microF and 500 V/cm, which resulted in luciferase activity up to 5000-fold above background 9-12 hr after electroporation. Constructs that contained the luciferase gene without amebic flanking sequences or that contained a simian virus 40 promoter, enhancer, and polyadenylylation signal produced only background levels of luciferase activity. The ability to introduce and express genes in amebae will now permit a genetic analysis of the virulence of this organism, which remains a serious threat to world health.

Animals↗

PubMed2002

Proteasome inhibitors reduce luciferase and beta-galactosidase activity in tissue culture cells.

Reporter enzymes are commonly used in cell biology to study transcriptional activity of genes. Recently, reporter enzymes in combination with compounds that inhibit proteasome function have been used to study the effect of blocking transcription factor degradation on gene activation. While investigating the effect of proteasome inhibition on steroid receptor activation of the mouse mammary tumor virus (MMTV) promoter, we found that treatment with proteasome inhibitors enhanced glucocorticoid activation of the promoter attached to a chloramphenicol acetyltransferase (CAT) reporter, but inhibited activation of MMTV attached to a firefly luciferase or beta-galactosidase reporter. MMTV RNA levels under these conditions correlated with the promoter activity observed using the CAT reporter, suggesting that proteasome inhibitor treatment interfered with luciferase or beta-galactosidase reporter assays. Washout experiments demonstrated that the majority of luciferase activity was lost if the proteasome inhibitor was added at the same time luciferase was produced, not once the functional protein was made, suggesting that proteasome inhibition interferes with production of luciferase protein. Indeed, we found that proteasome inhibitor treatment dramatically reduced the levels of luciferase and beta-galactosidase protein produced, as determined by Western blot. Thus, treatment with proteasome inhibitors interferes with luciferase and beta-galactosidase reporter assays, possibly by inhibiting production of a functional reporter protein.

Cysteine Endopeptidases↗

PubMed2003

Cloning and expression of cDNA for a luciferase from the marine copepod Metridia longa. A novel secreted bioluminescent reporter enzyme.

Metridia longa is a marine copepod from which a blue bioluminescence originates as a secretion from epidermal glands in response to various stimuli. We demonstrate that Metridia luciferase is specific for coelenterazine to produce blue light (lambda(max) = 480 nm). Using an expression cDNA library and functional screening, we cloned and sequenced the cDNA encoding the Metridia luciferase. The cDNA is an 897-bp fragment with a 656-bp open reading frame, which encodes a 219-amino acid polypeptide with a molecular weight of 23,885. The polypeptide contains an N-terminal signal peptide of 17 amino acid residues for secretion. On expression of the Metridia luciferase gene in mammalian Chinese hamster ovary cells the luciferase is detected in the culture medium confirming the existence of a naturally occurring signal peptide for secretion in the cloned luciferase. The novel secreted luciferase was tested in a practical assay application in which the activity of A2a and NPY2 G-protein-coupled receptors was detected. These results clearly suggest that the secreted Metridia luciferase is well suited as a reporter for monitoring gene expression and, in particular, for the development of novel ultrahigh throughput screening technologies.

Amino Acid Sequence↗

PubMed1993

Gene therapy by intramuscular injection of plasmid DNA: studies on firefly luciferase gene expression in mice.

Direct injection of nonviral, covalently closed circular plasmid DNA into muscle results in expression of the DNA in myofiber cells. We have examined the expression of firefly luciferase DNA constructs injected into adult murine skeletal muscle. Considerable variation in luciferase enzyme expression was noted among constructs with different regulatory elements, among different batches of the same DNA construct, and among similar transfection experiments performed at different times. This variation was minimized by using single batches of plasmid DNA and by performing comparable sets of experiments concurrently. A quantitative experimental protocol was defined for comparing various aspects of the transfection process. We report that a luciferase construct containing the human cytomegalovirus immediate-early gene promoter plus intron A (a construct termed "p-CMVint-lux") showed the highest expression among several constructs tested. Dose-response and time course analyses of p-CMVint-lux DNA injections showed that maximal luciferase expression was achieved with 25 micrograms of DNA at 7-14 days post-injection. Selected manipulations of the transfection process were examined for their influence on luciferase expression. Variations in the rate of DNA injection, needle size, injection volume, and vehicle temperature had no significant effect on luciferase expression. The presence of endotoxin, cationic peptide, muscle stimulants or relaxants, vasoconstrictors, metal chelators, or lysosomal lytic reagents had no significant effect on expression. However, linearization of the DNA, injection of the DNA in water rather than saline, or inclusion of a DNA intercalating agent nearly abolished luciferase expression. And finally, increasing the injection dose by giving multiple injections over a 10-day period increased expression proportionally to the number of injections.

Amino Acid Sequence↗

PubMed2000

Refolding of firefly luciferase immobilized on agarose beads.

The renaturation yield of the denatured firefly luciferase decreased strongly with increasing protein concentration in a renaturation buffer, because of aggregation. In this study, firefly luciferase was immobilized on agarose beads at a high concentration. Although the protein concentration was extremely high (about 100-fold) compared to that of soluble luciferase, the renaturation yield was comparable with that for the soluble one. Thus, immobilization was shown to be effective for avoiding aggregation of firefly luciferase. It was also shown that the optimum buffer conditions for renaturation of the immobilized luciferase were the same as those for the renaturation in solution. Also, it was indicated that electrostatic interactions between a protein and the matrix have a negative effect on renaturation of the immobilized luciferase since the renaturation yield decreased at acidic pH only for the immobilized luciferase. These novel observations are described in detail in this paper.

Animals↗

PubMed2006

Consensus guided mutagenesis of Renilla luciferase yields enhanced stability and light output.

Luciferases, which have seen expansive employment as reporter genes in biological research, could also be used in applications where the protein itself is conjugated to ligands to create probes that are appropriate for use in small animal imaging. As the bioluminescence activity of commonly used luciferases is too labile in serum to permit this application, specific mutations of Renilla luciferase, selected using a consensus sequence driven strategy, were screened for their ability to confer stability of activity in serum as well as their light output. Using this information, a total of eight favorable mutations were combined to generate a mutant Renilla luciferase (RLuc8) that, compared with the parental enzyme, is 200-fold more resistant to inactivation in murine serum and exhibits a 4-fold improvement in light output. Results of the mutational analysis were also used to generate a double mutant optimized for use as a reporter gene. The double mutant had half the resistance to inactivation in serum of the native enzyme while yielding a 5-fold improvement in light output. These variants of Renilla luciferase, which exhibit significantly improved properties compared with the native enzyme, will allow enhanced sensitivity in existing luciferase-based assays as well as enable the development of novel probes labeled with the luciferase protein.

Amino Acid Sequence↗

PubMed2004

The characterization and hormonal regulation of kidney androgen-regulated protein (Kap)-luciferase transgenic mice.

The androgen-dependent regulation for the gene encoding the kidney androgen regulated protein (Kap) was examined in transgenic mice expressing luciferase (luc) under the control of the murine Kap promoter. Biophotonic imaging was used to visualize luciferase expression from the kidneys and various organs that was confirmed using luminometer assays. Kap-luc expression was observed at high levels in kidneys, epididymides, testes, and seminal vesicles in male mice, and in kidneys, ovaries, and uterus in female mice. Kap-luc expression was modulated by androgen and anti-androgen treatment in both male and female mice. Male mice were treated daily with the anti-androgenic compounds, cyproterone acetate (50 and 100 mg/kg/day) and flutamide (50 and 100 mg/kg/day), or vehicle for 16 days. Endpoints evaluated included in vivo biophotonic imaging, body weights, organ weights (liver, kidney, testes, epididymides, preputial gland, and seminal vesicles), protein luciferase assays and Western blot analysis. Biophotonic imaging was used to follow Kap-luc expression from each animal throughout the experiment using a sensitive imaging system. These imaging results correlated well with Western blot analysis and traditional endpoints of body and organ weights. Following treatment with anti-androgens, the luciferase signal was found to significantly decrease in the intact male mouse using in vivo biophotonic imaging and correlated with measurements of luciferase activity in homogenized organ extracts. The decrease in epididymal and seminal vesicle weight confirmed the action of the anti-androgens. In vivo imaging documented significant changes in luciferase expression within the first few days of the experiment indicative of the anti-androgenic activity of the drugs. Testosterone treatment significantly increased the Kap-luc bioluminescent signal in female mice. This increased luciferase induction was shown to be inhibited by coadministration of cyproterone (100 mg/kg/day). Our results indicate that biophotonic imaging may provide a useful approach for noninvasively tracking the effects of endocrine disruptors in specific tissues.

Androgen Antagonists↗

PubMed1993

Fluorescent polyene aliphatics as spectroscopic and mechanistic probes for bacterial luciferase: evidence against carbonyl product from aldehyde as the primary excited species.

The fluorescent alpha-parinaric acid (alpha-PAC) and beta-parinaric acid (beta-PAC) were converted to the corresponding aldehydes and alcohols all of which exhibited absorption and fluorescence properties closely resembling those of the parent acids. alpha-PAC and beta-PAC each binds to luciferase in competition with aldehyde. The hydrophobic nature of the aldehyde site was indicated by the enhanced fluorescence quantum yields of the bound alpha-PAC and beta-PAC. These two polyene acids and the beta-parinaryl alcohol were shown to stabilize the luciferase flavin-peroxide intermediate. alpha-Parinaraldehyde (alpha-PAD) and beta-parainaraldehyde (beta-PAD) were active substrates for Vibrio harveyi and Vibrio fischeri luciferases and, for the former enzyme, exhibited Km values similar to and quantum yields about 20-30% as those for decanal and dodecanal. For the V. harveyi luciferase with reduced FMN as a co-substrate, the alpha-PAD- or beta-PAD-initiated luminescence was indistinguishable from the normal emission obtained with octanal (lambda max 495 nm) showing no additional 430-nm component correlatable with emission from excited alpha-PAC or beta-PAC. In reactions using reduced 2-thioFMN for V. harveyi luciferase or reduced FMN for V. fischeri luciferase plus yellow fluorescent protein, the replacement of octanal by beta-PAD again resulted in no additional 430-nm emission. The lack of any emission correlatable with excited alpha-PAC, beta-PAC, or equivalent carbonyl product was not due to the quenching of the polyene moiety by chemical transformation, binding to luciferase, or a 100% energy transfer to the flavin 4a-hydroxide emitter.(ABSTRACT TRUNCATED AT 250 WORDS)

Aldehydes↗

PubMed1981

Luciferase inactivation in the luminous marine bacterium Vibrio harveyi.

Luciferase was rapidly inactivated in stationary-phase cultures of the wild type of the luminous marine bacterium Vibrio harveyi, but was stable in stationary-phase cultures of mutants of V. harveyi that are nonluminous without exogenous aldehyde, termed the aldehyde-deficient mutants. The inactivation in the wild type was halted by cell lysis and was slowed or stopped by O2 deprivation or by addition of KCN and NaF or of chloramphenicol. If KCN and NaF or chloramphenicol were added to a culture before the onset of luciferase inactivation, then luciferase inactivation did not occur. However, if these inhibitors were added after the onset of luciferase inactivation, then luciferase inactivation continued for about 2 to 3 h before the inactivation process stopped. The onset of luciferase inactivation in early stationary-phase cultures of wild-type cell coincided with a slight drop in the intracellular adenosine 5'-triphosphate (ATP) level from a relatively constant log-phase value of 20 pmol of ATP per microgram of soluble cell protein. Addition of KCN and NaF to a culture shortly after this drop in ATP caused a rapid decrease in the ATP level to about 4 pmol of ATP per microgram whereas chloramphenicol added at this same time caused a transient increase in ATP level to about 25 pmol/microgram. The aldehyde-deficient mutant (M17) showed a relatively constant log-phase ATP level identical with that of the wild-type cells, but rather than decreasing in early stationary phase, the ATP level increased to a value twice that in log-phase cells. We suggest that the inactivation of luciferase is dependent on the synthesis of some factor which is produced during stationary phase and is itself unstable, and whose synthesis is blocked by chloramphenicol or cyanide plus fluoride.

Adenosine Triphosphate↗

PubMed1998

Modulation of human immunodeficiency virus type 1-induced syncytium formation by the conformational state of LFA-1 determined by a new luciferase-based syncytium quantitative assay.

The ICAM-1/LFA-1 interaction has been clearly demonstrated to play an active role in syncytium formation induced by human immunodeficiency virus type 1 (HIV-1). Since it is known that a high-affinity state of LFA-1 for ICAM-1 can be induced through conformational change, such a high-affinity state may also contribute to the process of syncytium formation. In this study, we have investigated the involvement of the conformational status of LFA-1 in HIV-1-dependent syncytium formation by using the anti-LFA-1 antibody NKI-L16, which is known to activate the high-affinity state. Initial visual observations by light microscopy indeed suggested that the addition of the NKI-L16 antibody led to bigger and more numerous syncytia when different cell lines were tested. To further analyze this NKI-L16-dependent increment of syncytium formation in a quantitative assay, a new luciferase-based assay was developed by using a T-cell line containing an HIV-1 long terminal repeat (LTR)-driven luciferase construct (1G5) in coincubation with an HIV-1-positive cell line (J1.1). Upon fusion, the viral Tat protein could diffuse to the 1G5 cells, leading to a transcriptional increase of the HIV-1 LTR-driven luciferase gene. Initial evaluation of this assay showed a good correlation between the level of syncytium formation determined by microscopic observation and the level of measured luciferase activity. In addition, this assay showed a greater induction of enzymatic activity correlating with syncytium formation in comparison to a similar incubation with the HeLa-CD4-LTR-beta-gal indicator cell line. By using this test, NKI-L16 treatment of 1G5/J1.1 cells led to a three- to sevenfold increase in HIV-1 LTR-driven luciferase activity. The syncytium-dependent luciferase activity in NKI-L16-treated cells could be blocked by classical syncytium inhibitors such as soluble CD4, anti-CD4, and anti-gp120 antibodies. Inhibition could also be observed with specific blocking agents for the chemokine receptor CXCR4, as well as with soluble ICAM-1, anti-LFA-1, anti-ICAM-1, and anti-ICAM-2 blocking antibodies, indicating the requirement for the LFA-1/ICAM interaction. Treatment of peripheral blood mononuclear cells with NKI-L16 resulted in a higher level of syncytium formation in the presence of the cell line J1.1. Conversely, when PBMCs were infected with two different syncytium-inducing HIV-1 primary isolates, coincubation with NKI-L16-pretreated 1G5 cells led to higher levels of luciferase activity for both virus isolates. Our results therefore show for the first time a direct role for the LFA-1 high-affinity state in virus-mediated syncytium formation. Based on the demonstration that an increase in ICAM-1 binding is induced by T-cell activation, these data suggest an in vivo involvement of the high-affinity state of LFA-1 in HIV-1-induced syncytium formation. Moreover, syncytia might preferentially occur in lymph nodes, since this microenvironment harbors a high proportion of activated T cells.

Antibodies↗

PubMed1995

Virus-mediated expression of firefly luciferase in the parasitic protozoan Giardia lamblia.

Giardia lamblia, a prevalent human pathogen and one of the lineages that branched earliest from prokaryotes, can be infected with a double-stranded RNA virus, giardiavirus (GLV). The 6,277-bp viral genome has been previously cloned (A.L. Wang, H.-M. Yang, K.A. Shen, and C.C. Wang, Proc. Natl. Acad. Sci. USA 90:8595-8599, 1993; C.-H. Wu, C.C. Wang, H.M. Yang, and A.L. Wang, Gene, in press) and was converted to a transfection vector for G. lamblia in the present study. By flanking the firefly luciferase gene with the 5' and 3' untranslated regions (UTRs) of the GLV genome, transcript of the construct was synthesized in vitro with T7 polymerase and used to transfect G. lamblia WB trophozoites already infected with GLV (WBI). Optimal electroporation conditions used for the transfection were set at 1,000 V/cm and 500 microF, which resulted in expression of significant luciferase activity up to 120 h after electroporation. Furthermore, the mRNA and the antisense RNA of the luciferase gene were both detected by reverse transcription and PCR from 6 to 120 h postelectroporation, whereas no antisense RNA of luciferase was observed in the electroporated virus-free Giardia WB trophozoites. The mRNA of luciferase was detectable in the virus-free trophozoites by reverse transcription and PCR only up to 20 h after the electroporation, indicating that the introduced mRNA was replicated only by the viral RNA-dependent RNA polymerase inside the WBI cells. This expression of luciferase was dependent on the presence of UTRs on both ends of the viral genome transcript, including a putative packaging site that was apparently indispensable for luciferase expression. This is the first time that a viral vector in the form of mRNA URTs has been successfully used in transfecting a protozoan.

Animals↗

PubMed2003

A dual luciferase multiplexed high-throughput screening platform for protein-protein interactions.

To study the biology of regulators of G-protein signaling (RGS) proteins and to facilitate the identification of small molecule modulators of RGS proteins, the authors recently developed an advanced yeast 2-hybrid (YTH) assay format for GalphaZ and RGS-Z1. Moreover, they describe the development of a multiplexed luciferase-based assay that has been successfully adapted to screen large numbers of small molecule modulators of protein-protein interactions. They generated and evaluated 2 different luciferase reporter gene systems for YTH interactions, a Gal4 responsive firefly luciferase reporter gene and a Gal4 responsive Renilla luciferase reporter gene. Both the firefly and Renilla luciferase reporter genes demonstrated a 40- to 50-fold increase in luminescence in strains expressing interacting YTH fusion proteins versus negative control strains. Because the firefly and Renilla luciferase proteins have different substrate specificity, the assays were multiplexed. The multiplexed luciferase-based YTH platform adds speed, sensitivity, simplicity, quantification, and efficiency to YTH high-throughput applications and therefore greatly facilitates the identification of small molecule modulators of protein-protein interactions as tools or potential leads for drug discovery efforts.

Dose-Response Relationship, Drug↗

PubMed2003

The p53-inhibitor pifithrin-alpha inhibits firefly luciferase activity in vivo and in vitro.

BACKGROUND: Pifithrin-alpha is a small molecule inhibitor of p53 transcriptional activity. It has been proposed that the use of pifithrin-alpha in conjunction with chemotherapeutic and radiation therapies for cancer will reduce the side effects of these treatments in normal tissue that still contains wild type p53. In addition, pifithrin-alpha provides a useful tool in the laboratory to investigate the function of p53 in model systems. RESULTS: While investigating the effects of pifithrin-alpha on the transcriptional activity of NF-kappaB, we observed a strong inhibition of reporter plasmids containing the firefly luciferase gene. Firefly luciferase is one of the most commonly used enzymes in reporter gene assays. In contrast, no inhibition of reporter plasmids containing Renilla luciferase or chloramphenicol acetyltransferase was observed. The inhibition of firefly luciferase activity by pifithrin-alpha was observed both in vivo and in vitro. Pifithrin-alpha did not inhibit firefly luciferase protein expression, but rather suppressed light production/emission, since addition of exogenous pifithrin-alpha to active extracts inhibited this activity. Furthermore, pifithrin-alpha also inhibited recombinant firefly luciferase protein activity. CONCLUSIONS: Among its other biological activities, pifithrin-alpha is an inhibitor of firefly luciferase activity. Caution must therefore be taken when using this compound, which has been characterised as an inhibitor of p53 transcriptional activity, to investigate effects on gene expression using transiently transfected reporter plasmids. Furthermore, these results demonstrate that when using novel compounds, the choice of vectors used in the experimental procedures might be of great importance for the correct conclusions to be made.

Animals↗

PubMed2006

Catalytic properties of domain-exchanged chimeric proteins between firefly luciferase and Drosophila fatty Acyl-CoA synthetase CG6178.

Firefly luciferase and fatty acyl-CoA synthetase are members of the acyl-CoA synthetase super family, which consists of a large N-terminal domain and a small C-terminal domain. Previously we found that firefly luciferase has fatty acyl-CoA synthetic activity, and also identified that the homolog of firefly luciferase in Drosophila melanogaster (CG6178) is a fatty acyl-CoA synthetase and is not a luciferase. In this study, we constructed chimeric proteins by exchanging the domain between Photinus pyralis luciferase (PpLase) and Drosophila CG6178, and determined luminescence and fatty acyl-CoA synthetic activities. A chimeric protein with the N-terminal domain of PpLase and the C-terminal domain of CG6178 (Pp/Dm) had luminescence activity, showing approximately 4% of the activity of wild-type luciferase. The Pp/Dm protein also had fatty acyl-CoA synthetic activity and the substrate specificity was similar to PpLase. In contrast, a chimeric protein with the N-terminal domain of CG6178 and the C-terminal of PpLase (Dm/Pp) had only fatty acyl-CoA synthetase activity, and the substrate specificity was similar to CG6178. These results suggest that the N-terminal domain of firefly luciferase is essential for substrate recognition, and that the C-terminal domain is indispensable but not specialized for the luminescence reaction.

Adenine↗

PubMed2000

Pitfall of an internal control plasmid: response of Renilla luciferase (pRL-TK) plasmid to dihydrotestosterone and dexamethasone.

The thymidine kinase promoter-Renilla luciferase reporter plasmid (pRL-TK) is commonly used as a control for transfection efficiency in the Dual-Luciferase Reporter Assay System. While investigating hormone response elements in the promoters of the androgen-dependent, epididymis-specific EP2 gene, we found that hormone treatment affected the luciferase activity of pRL-TK-transfected cells. In African Green Monkey Kidney (CV-1) cells, cotransfected transiently with a hormone-responsive promoter-firefly luciferase reporter plasmid and with pRL-TK, Renilla luciferase activity increased in response to dihydrotestosterone (DHT) and decreased in response to dexamethasone (DEX). When a thromboxane synthase promoter Renilla luciferase plasmid (pRL-TS) was used in place of pRL-TK, Renilla luciferase activity remained constant in CV-1 cells treated with DHT but decreased in CV-1 cells treated with DEX. In transfection studies, internal control plasmid expression in response to treatment must be carefully monitored to ensure proper interpretation of normalized results.

Animals↗

PubMed1996

Dexamethasone stimulates luciferase gene expression through the rat bone sialoprotein gene promoter in transgenic mice.

Bone sialoprotein (BSP) is expressed by differentiated osteoblasts during the initial formation and mineralization of bone matrix. Studies using transgenic mice harboring 2.7 kb of the rat BSP promoter linked to a luciferase reporter gene have shown luciferase activity in bone and other mineralized tissues while most soft tissues tested expressed a much lower level of the reporter gene. To study regulation of the transgene, mice were administered dexamethasone (dex) by intramuscular injection. After 4 h and 24 h, various tissues were dissected from the treated mice as well as from untreated transgenic littermates. Luciferase assays showed that dex stimulated expression of the transgene significantly. In bone tissues, dex increased the average luciferase activity 1.6- to 11-fold compared with control tissues from untreated transgenic mice. The luciferase activity in lung, liver and kidney remained at a low level and showed no increase with dex treatment. In some animals, however, the luciferase activity in brain and skin was also increased after dex administration. These experiments indicate that a transgene comprising 2.7 kb of the rat BSP promoter linked to a luciferase reporter is regulated in a tissue and developmental stage-dependent manner and that glucocorticoid-induced stimulation of BSP gene expression may be mediated within this region of the promoter.

Animals↗