Luciferase reporter gene detection kit catalog number luc1 storage temperature 70 c technical bulletin product description firefly luciferase is one of the most commonly utilized reporter genes for the study of gene expression. The onestep luciferase assay system is designed to be used for highthroughput, sensitive. Stimulation of transcription of the genes for luciferase lux a and lux b was required for efficient synethesis of luciferase. The luciferase gene or luciferase bioluminescence does not affect the growth of tumors in mice. A specific gene promoter can be attached to the dna that codes for firefly luciferase and introduced into an organism. Luciferase assays provide high sensitivity and timeresolution for measuring promoter activity. Dualluciferase imaging in vivo promega connections.
By cloning the 3utr of tet2 containing these sites downstream of the firefly luciferase gene in the pmirreport reporter vector, the resultant plasmid was able to be cotransfected with a mimic of mir29 or a scrambled mirna control. The use of the luciferase reporter system for in planta. If the luminometer is not connected to a printer or. Imanis offers a wide range of highquality, readytouse reporter gene cell lines that can be used to monitor tumors in living animals through noninvasive imaging. The length of the enhancer element differs for each luciferase reporter vector, therefore the positions indicated on the vector map should be adjusted accordingly.
Aug 26, 20 the luciferase reporter assay is commonly used as a tool to study gene expression at the transcriptional level. Other organisms, including plants, that express the luciferase luc gene will also glow faintly green when supplied with luciferin. In the dlr assay, the activities of firefly photinus pyralis and renilla renilla reniformis, also known as sea pansy luciferases are measured sequentially from a single sample. The luciferase reporter genes are expressed downstream of the target. Apr 14, 2014 the authors designed a firefly luciferase reporter for kinase activity of a tgfbeta1 receptor and a nanoluc luciferase based reporter for tgfbetadependent transcription and were able to monitor the effect of tgfbeta or specific inhibitors on either receptor activation firefly activity or gene expression nanoluc activity. Luciferase definition of luciferase by the free dictionary. In this article, we focus on two luciferase reporters, firefly and renilla, the development of recombinant proteins and their use in dualreporter assays, since much of the luciferase reporter gene technology was developed by promega. Luciferase assays boeckx, 1984 have become the assay of choice in recent years because. The luciferase gene was isolated from the pgemluc vector in a similar fashion. Imaging luciferaseexpressing viruses research nebraska. However, this was recently changed with the introduction of fungal. Localization of luciferase protein in photocytes of firefly lantern, as well as in animal and plant cells, indicated that the. Luciferase reporter gene assay, high sensitivity sigmaaldrich. Promega quantilum recombinant luciferase fisher scientific.
The oneglo luciferase assay buffer and oneglo luciferase assay substrate, provided with this system, are combined to form the oneglo reagent see section 3. Reporter gene expression for monitoring gene transfer. The assay measures dynamic changes in proteinprotein interactions and can be used for both cell culture and whole animals. Although they share a common name, many luciferases do not share sequence homology. In photinus the luciferase is encoded by a transcript of about 1,800 nt, which is synthesized from a single copy luc gene containing six introns dewet et al.
Luciferase based reporter gene assays are widely used because of their ultrasensitive detection capacity and wide dynamic range. Thus, the luciferase gene can be used to report the activity of a promoter for another gene. Genes for the luciferase enzyme of vibrio harveyi were isolated in escherichia coli by a general method in which nonluminous, transposon insertion mutants were used. It is widely used because it is convenient, relatively inexpensive, and gives quantitative measurements instantaneously. When using the microplate format, black or white microplates must be used we recommend using black microplates. An enzyme present in the cells of bioluminescent organisms that catalyzes the oxidation of luciferin and atp, producing light. Mice received biweekly injections of either pbs or dluciferin 150 mgkg and. Firefly luciferase is a member of a family of enzymes that emit visible light in the course of catalyzing substrate conversion to product.
Their primary uses are for mapping the control regions of genes, for measuring signal transduction pathways modulated by hormones or disease, as a tool in gene therapy and drug discovery, and for noninvasive wholeanimal. Luciferase expression and bioluminescence does not affect. The scientist produces a construct in which the regulatory region of a target gene is fused with the dna coding sequence for luciferase figure 15. Read on luminometer in the gel room using promega luciferase assay substrate thawed and mixed at rt. The nucleotide sequence of the luciferase gene from the firefly photinus pyralis was determined from the analysis of cdna and genomic clones. The luciferase reporter gene assay, based on the responsibleelementregulated expression of luciferase, has been widely applied in the highthroughput functional assay for. Jan 27, 2015 the luciferase reporter gene assay, based on the responsibleelementregulated expression of luciferase, has been widely applied in the highthroughput functional assay for many targets because of. William kaelins lab contains the insert firefly luciferase and is published in proc natl acad sci u s a. Given that luciferase requires a special camera and optical filter such as a gel documentation system, xgal is ideal because it may be screened by eye. Assay lysates for luciferase activity and protein concentration. Firefly luciferase complementation imaging assay for.
These assays involve placing a genetic regulatory element upstream of a luciferase gene and then transferring the resulting reporter construct into animal cells, plant cells or bacteria through transfection. Several renillabased control reporter genes are commercially available. Both words are derived from the latin word lucifer meaning lightbringer. Allard and kevin kopish, promega corporation luciferase reporter technology gives the researcher unparalleled sensitivity, dynamic range, versatility and ease of use when investigating questions that involve gene regulation. All of the luciferase vectors contain the panomics signature sequence. The luciferase reporter assay is commonly used as a tool to study gene expression at the transcriptional level. The luciferinluciferase reaction is actually an enzyme. Firefly luciferase as a reporter of regulated gene. The glow is widely used as an assay for luc expression, which acts as a reporter for the activity of any genetic regulation that controls the expression of its gene. While the gus reporter is a valuable reporter for visualizing the evenness of expression across the.
A firefly luciferase dual color bioluminescence reporter assay. Luciferase imaging has been used to study the location of infection by replicationdefective and replicationcompetent viruses and to track changes in the distribution of viruses in mouse models. Publish by abstract the use of the luciferase reporter system for in planta gene expression studies wessel van leeuwen1, marc j. Microrna29 negatively regulates expression of oncogene tet2. Luciferase definition of luciferase by medical dictionary. Optical imaging of luciferage gene expression has become a powerful tool to track cells and viruses in vivo in small animal models. Dualluciferase reporter assay system technical manual no. H 2 o 2 repressed luciferase activity, whereas heavy metals, such as cdcl 2, and other hormones, including 2,4d and salicylate, had no apparent effect on the expression of the luc reporter gene fig. Firefly luciferase as a reporter of regulated gene expression. Luciferase reporter gene detection kit luc1 technical. It is an extremely sensitive, rapid, and easy to use procedure. The luciferase reporter gene assay, high sensitivity can be performed in all automated or manual luminometers in tube and microplate formates as well as in scintillation counters, or using photographic films. A separate dna construct encodes the protein hypothesized to affect transcription. Luciferase is an enzyme used for bioluminescence by various organisms in nature, most famously the firefly.
The dualluciferase reporter dlr assay system provides an efficient means of performing dualreporter assays. Pdf luciferase as a reporter of gene activity in plants. If the luminometer is not connected to a printer or computer, record. Use biorad assay for protein concentrations in 20ul of.
This kit is a new and improved replacement for the firefly luciferase assay kit catalog no. Screening for gene regulation mutants by bioluminescence. The behaviours of the transfected cells were evaluated by a fireflyluciferase reporter gene harbouring the promoter region of i. Our genetic screen uses the firefly luciferase reporter gene driven by the cold, drought, salt, and abscisic acid abaresponsive rd29a promoter rd29aluc. Gel purification was conducted in order to isolate the. Luciferase reporter gene assays monitor the transcription of specific genes in cells and in vivo through the detection of light generated by the enzyme luciferase. Furthermore, the sensitivity of the luciferase assay in extracts of transformed plant tissue makes it a particularly useful marker in transient or stable transformation experiments. It has broad applications across various fields of cell and molecular biology wherever you want to measure or. Promega quantilum recombinant luciferase quantilum recombinant luciferase is a luciferase expressed from a cloned gene from the north american firefly photinus pyralis. Description genetic reporters are used commonly in cell biology to study. Firefly luciferase is the ideal reporter gene for monitoring circadian rhythms in eukaryotes.
Cauliflower mosaic virus 35s promoter, luciferase reporter system, petunia. Luciferase article about luciferase by the free dictionary. Renilla luciferase assay system quick protocol, 9fb066 promega. Luciferase definition is an enzyme that catalyzes the oxidation of luciferin. Screening for gene regulation mutants by bioluminescence imaging. Luciferase reporter gene assay, high sensitivity y version 20. The readytoglow secreted luciferase reporter assay includes a promoterless plasmid and is designed to insert your promoter of interest upstream of the sequenceoptimized metridia secreted luciferase reporter gene. For luciferase activity, pipet 20ul of each lysate into a 5ml polystyrene falcon tube.
The present custom is to use generic names according to origine. Gene trapping with firefly luciferase in arabidopsis. This firefly luciferase assay kit is designed for simple and efficient quantitation of firefly luciferase reporter enzyme activity from cultured cells with high sensitivity and linearity. The use of the luciferase reporter system for in planta gene. Luciferase definition of luciferase by merriamwebster. The 5 end of the luciferase mrna was determined by both s1 nuclease analysis and primer extension. The authors designed a firefly luciferase reporter for kinase activity of a tgfbeta1 receptor and a nanoluc luciferasebased reporter for tgfbetadependent transcription and were able to monitor the effect of tgfbeta or specific inhibitors on either receptor activation firefly activity or gene expression nanoluc activity. Luciferase as a reporter of gene activity in plants. Luciferasebased reporter gene assays are widely used because of their ultrasensitive detection capacity and wide dynamic range. The gene contains six introns, all less than 60 bases in length. Localization of luciferase protein in photocytes of firefly lantern, as well as in animal and plant cells, indicated that the enzyme is targeted to peroxisomes.
The combined results give the prerequisites for the correct utilisation of the luciferase reporter system, especially for in vivo gene expression studies in plant research. If the luminometer is not connected to a printer or computer, record the reading. Using agrobacterium tumefaciensmediated transformation, 20,261 promoter trap arabidopsis lines were generated, carrying the tdna of the ptluc gene fusion vector fig. Here, we describe a reporter gene based strategy to screen for mutants affected in abiotic stressregulated gene transcription.
Recent advances in application of luciferase reporter genes are re viewed and documented by examples of in vivo visualization of their expression in transgenic. To call, write, fax, or email us, visit, and select your home country to. Mar 10, 2017 luciferase reporter gene assays monitor the transcription of specific genes in cells and in vivo through the detection of light generated by the enzyme luciferase. Firefly luciferase is widely used as a reporter for studying gene regulation and function. Jerry crabtrees lab contains the insert 3x nfat binding sequence and is published in nature.
Their primary uses are for mapping the control regions of genes, for measuring signal transduction pathways modulated by hormones or disease, as a tool in gene therapy and drug discovery, and for noninvasive wholeanimal imaging. Luciferase activity occurs only when the two fused proteins interact, resulting in reconstituted luciferase enzyme, which can be detected by luminometer or a low light imaging device. The luciferase from the firefly photinus pyralis ffluc is the most widely used among the luciferase family members as a reporter gene for molecular and cell biological. The scientist transfects a cell culture system, such. The luciferase assay system was developed for reporter quantitation in mammalian cells. Luciferase reportersreadytoglow secreted luciferase assay.
Nov 22, 2010 the luciferase gene or luciferase bioluminescence does not affect the growth of tumors in mice. The use of the luciferase reporter system for in planta gene expression studies plant mol. It is a very sensitive genetic reporter due to the lack. Luciferase is a generic term for the class of oxidative enzymes that produce bioluminescence, and is usually distinguished from a photoprotein. Amsbio offers new luciferase assay systems to help improve. The activity of the gene promoter can then be studied by measuring the bioluminescence produced in the luciferase reaction. Screening for in situ luciferase gene fusions and characterization of ptluctagged arabidopsis collection.
Bacterial and firefly luciferase genes in transgenic. Typically, the expression of a gene of interest is reported on with high sensitivity and. Bacterial and firefly luciferase genes in transgenic plants. We discuss factors in choosing a reporter vector, the advantages of bioluminescence and other uses of. Hagendoorn, tom ruttink, remco van poecke, linus h. The tagged gene thus appeared to be controlled by an abadependent regulatory pathway in response to osmotic stress. Nih3t3 cells expressing the luciferase gene from rous sarcoma virus were lysed with 1x cell culture lysis. Here, we describe a reporter genebased strategy to screen for mutants affected in abiotic stressregulated gene transcription. The firefly luciferase, assayedin vivo with a lowlight video camera, acts as a noninvasive, realtime reporter of the temporal and spatial regulation of gene expression in single plants. Reporter gene expression for monitoring gene transfer stephen welsh.
1402 362 1012 1155 1327 1411 423 292 113 377 429 302 687 1303 1168 32 274 679 897 139 532 1302 787 1006 1478 656 245 1275 390 574 962 1144 765