The most commonly occurring bioluminescence system in nature is the deepsea imidazolopyrazine bioluminescence system. Both words are derived from the latin word lucifer meaning. A highly sensitive proteinprotein interaction assay based. Bioglonl assay reagent contains a new luciferase substrate, resulting in a reagent that is brighter, more stable and more tolerant to sample. Geneencoding luciferase very often fulfils the function of the reporter gene, because this enzyme induces luminescence after oxidation of specific sub strates 69. The luciferase from the firefly photinus pyralis ffluc is the most widely used among the luciferase family members as a reporter gene for. William kaelins lab contains the insert firefly luciferase and is published in proc natl acad sci u s a. Generally, 100fold greater sensitivity can be achieved over the chloramphenicol acetyltransferase cat assay. Dual luciferase assay system for rapid assessment of gene. The enzyme catalyses the oxidation of firefly luciferin, requiring oxygen and atp. The fluc coding region displays cryptic promoter activity both in mammalian and yeast cells. Reporter gene systems based upon modified luciferase genes isolated from organisms, ranging from bacteria to insects, have proven to be important tools for plant molecular studies. Bacterial and firefly luciferase genes in transgenic plants. Typically, a reporter gene is cloned with a dna sequence of interest into an expression vector that is then transferred into cells.
The backbone of the pgl2 luciferase reporter vectors was redesigned for the pgl3 vectors for increased expression, with a modified coding region for. A new reporter system has been developed for quantifying gene expression in the yeast saccharomyces cerevisiae. The dualglo luciferase assay system is designed to work in growth media commonly used for mammalian cells with or without added serum. Pdf dual luminescencebased reporter gene assay for. 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. 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 luciferase assay system yields linear results over at least eight orders of magnitude.
The luciferase assay system was developed for reporter quantitation in mammalian cells. For example, an enzyme known as luciferase in fireflies produces light by acting on a molecule called luciferin. Fluc is the bestcharacterized bioluminescent protein and is used widely as a tool for reporting gene expression levels within living cells gelmini et al. A highly sensitive proteinprotein interaction assay based on. It has broad applications across various fields of cell and molecular biology wherever you want to measure or. The properties of the firefly luciferase luc make it a very good nondestructive reporter to quantify and image transgene promoter activity in plants. Us7723502b2 modified luciola cruciata luciferase gene and. The luciferase assay reagent generates light that is nearly constant for at least 1 minute and so the assay. Gaussia luciferase gluc and cypridina luciferase cluc are two naturally secreted luciferases with readytouse lentiviral expression particles for luciferaseproduct manual, page 6 of 12 extremely high sensitivities and can be used for realtime and continuous monitoring of gene.
The strategies used to design the synthetic luciferase transgenes could be applied to other transgenes that are aggressively silenced. Firefly luciferase gene contains a cryptic promoter. In addition, multigene plasmids were cloned, allowing the detection of all. An extremely sensitive reagent for rapid quantitation of firefly luciferase. The ability of the fulllength luciferase gene to activate cryptic or enhancerless promoters was also greatly reduced or eliminated by this 5 deletion. The 5 end of the luciferase mrna was determined by both s1 nuclease analysis and primer extension. Cloning of firefly luciferase cdna and the expression of. Because of the requirement of atp, firefly luciferases have been used extensively in. So, luciferase uses a very energetic process to create light. The luciferase gene from the firefly, photinus pyralis, was used as a reporter of gene expression by light production in transfected plant cells and transgenic plants. Introduction of the luciferase and other lightemitting proteins e. Building customizable autoluminescent luciferasebased.
A firefly luciferase dual color bioluminescence reporter. Scientists have identified the enzymes that drive several of these systems and used them to build reporters that can study the activity of genes in the tissues of plants and. 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. The system is a convenient alternative to firefly photinus pyralis reporter systems and is designed to yield reliable, linear results for a concentration range over 7 orders of magnitude. The invention discloses a kind of method utilizing luciferase reporter gene determination core promoter, comprise the following steps. The gene encoding renilla luciferase is fused to a constitutive promoter pgk1 or spt15 and integrated into the yeast genome at the. Download sds pdf 41 kb english united states certificate of analysis. This cdna contained sufficient coding information to direct the synthesis of active firefly luciferase in e. Unlike the chip or emsa assays, which only assess the ability of a protein to interact with a region of dna, a luciferase assay is able to establish a functional.
Indepth characterization of firefly luciferase as a reporter. The present invention provides a polynucleotide or polynucleotides encoding oplophorus luciferase which is composed of 19 kda and 35 kda proteins, or the 19 kda photoprotein, the recombinant secretional oplophorus luciferase or the 19 kda photoprotein encoded by the polynucleotides, an expression vector containing the polynucleotides and a host transformed with the vector. Recent advances in application of luciferase reporter genes are re viewed and documented by examples of in vivo visualization of their expression in transgenic. Transient and stable expression of the firefly luciferase. A complementary dna clone of the firefly luciferase gene under the control of a plant virus promoter cauliflower mosaic virus 35 s rna promoter was introduced into plant protoplast cells daucus carota by electroporation. To see articles that cite the use of the dualglo luciferase assay system, visit. The methods of the invention entail improved kinetics of light production from luciferase activity in beetle luciferase luciferin reactions. Firefly luciferase an overview sciencedirect topics. The destabilized and optimized luc2p luciferase gene offers greater sensitivity and shorter induction times in the reporter assay. The nucleotide sequence of the luciferase gene from the firefly photinus pyralis was determined from the analysis of cdna and genomic clones. Alternative luciferase for monitoring bacterial cells. Pdf luciferase gene of a caribbean fireworm syllidae. Pdf codonoptimized luciola italica luciferase variants.
Firefly luciferase is the lightemitting enzyme responsible for the bioluminescence of fireflies and click beetles. The bioglonl luciferase assay system provides a highly sensitive, robust and homogeneous reagent for the detection of nanoluc luciferase reporter gene expression in promega bioassays. The system relies on two different reporter genes, renilla and firefly luciferase, to evaluate regulated gene expression. Luciferase is an enzyme used for bioluminescence by various organisms in nature, most famously the firefly. The pgl3 luciferase reporter vectors provide a basis for the quantitative analysis of factors that potentially regulate mammalian gene expression. The luciferase reporter gene is a useful tool for determining the efficacy of transfection of plasmid dna and adenovirusmediated gene transfer in vivo. Both words are derived from the latin word lucifer meaning lightbringer. Amino acid sequences and expression analysis of luciferase candidate genes. Stable transfection of the chimeric gal4 estrogen receptor and luciferase gene constructs in mcf7 breast cancer and hepa. To address this, we explored whether a transposonbased system could identify sites in the viral genome where endogenous. The gene contains six introns, all less than 60 bases in length. Pdf luciferase gene of a caribbean fireworm syllidae from. Novel gene encoding a unique luciferase from the fireworm. Therapeutic transgene expression from oncolytic viruses represents one approach to increasing the effectiveness of these agents as cancer therapeutics.
A separate dna construct encodes the protein hypothesized to affect transcription. Using the wellcharacterized luciferase gene of the japanese syllid odontosyllis undecimdonta as a reference, a complete. Pdf nanoluc reporter for dual luciferase imaging in living. Expression of the luciferase gene fusion product confers on the host the ability to glow in the dark. The different luc2 genes code for luciferase proteins with different stabilities.
Stimulation of transcription of the genes for luciferase lux a and lux b was required for efficient synethesis of luciferase. Identification of novel insertion sites in the ad5 genome. Luciferasebased reporter gene assays are widely used because of their ultrasensitive detection capacity and wide dynamic range. Two types of luciferase genes, cloned from bacteria and firefly, are used as sensitive reporter systems in a wide variety of. The availability of cloned luciferase genes from fireflies luc and from bacteria luxab has led to the widespread use of bioluminescence as a reporter to measure cell viability and gene expression. In the case of the oncolytic adenovirus ad, however, the genomic packaging capacity is constrained.
Pdf codonoptimized luciola italica luciferase variants for. The use of the luciferase reporter system for in planta gene. A firefly luciferase fluc counts among the most popular reporters of presentday molecular and cellular biology. Typically, a promoter region containing 2 to 3 kb of a 5. The firefly luciferase gene luc from photinus pyralis is a new tool for. The scientist transfects a cell culture system, such.
The methods of the invention entail improved kinetics of light production from luciferase activity in beetle luciferaseluciferin reactions. Effective methods for monitoring gene expression and regulation based on bioluminescence bl the emission of light by living organisms are well known 1,2,3. Alternative luciferase for monitoring bacterial cells under. Pdf biotechnological advances in luciferase enzymes. For an overview and information on the development of the dualglo luciferase assay system, see sections 6.
Promoterless firefly luciferase basic vectors promega corporation. The plasmid containing the lux gene is isolated from the e. Gene encoding luciferase very often fulfils the function of the reporter gene, because this enzyme induces luminescence after oxidation of specific sub strates 69. Although they share a common name, many luciferases do not share sequence homology. When this oxygenated luciferin breaks, forming carbon dioxide in the process, it leaves behind a highly excited form that then emits the light. 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. Aug 24, 2018 amino acid sequences and expression analysis of luciferase candidate genes. Firefly luciferase is a member of a family of enzymes that emit visible light in the course of catalyzing substrate conversion to product.
Many animals have evolved the capacity to produce light from chemical reactions. The primary focus is placed on firefly luciferase fluc. The luciferase assay is a very sensitive and convenient way to examine the transcriptional activity of a gene. Readytouse lentiviral particles for luciferase expression. Nanoluc reporter for dual luciferase imaging in living animals. In circadian research, fluc is frequently expressed under the control of circadian regulated promoter sequences to report oscillations in transcriptional activity, for example, per2. Pdf synthetic versions of firefly luciferase and renilla luciferase.
Development of a cytotoxicity assay based on transient expression of different luciferases in cytosol. Using the wellcharacterized luciferase gene of the japanese syllid odontosyllis undecimdonta as a reference, a complete bestmatching luciferase open reading frame. Apr 16, 2018 effective methods for monitoring gene expression and regulation based on bioluminescence bl the emission of light by living organisms are well known 1,2,3. The luciferase reporter assay is commonly used as a tool to study gene expression at the transcriptional level. Renilla luciferase assay system is designed to provide a fast, sensitive method of detecting luciferase from sea pansy renilla reniformis. Cn103382505b luciferase reporter gene is utilized to. Development and characterization of a novel luciferase based. Pdf luciferase as a reporter of gene activity in plants. It has a cofactor, termed a luciferin, that forms a highly strained complex with oxygen, using an atp molecule to help set everything up. Development of a stably transfected estrogen receptor. Us7125697b2 us10377,714 us37771403a us7125697b2 us 7125697 b2 us7125697 b2 us 7125697b2 us 37771403 a us37771403 a us 37771403a us 7125697 b2 us7125697 b2 us 7125697b2 authority us united states prior art keywords protein luciferase polynucleotide kda seq id prior art date 20000426 legal status the legal status is an assumption and is not a.
Us7723502b2 modified luciola cruciata luciferase gene. Luciferase is a generic term for the class of oxidative enzymes that produce bioluminescence, and is usually distinguished from a photoprotein. A complementary dna clone of the firefly luciferase gene under the control of a plant virus promoter cauliflower mosaic virus 35 s rna promoter was introduced into plant protoplast. Development and characterization of a novel luciferase. This polypeptide comigrated with luciferase on nadodso4page and produced bioluminescence upon the addition of luciferin and atp.
Haemoglobin interferes with the ex vivo luciferase. There are currently no references for firefly luciferase antibody. Indepth characterization of firefly luciferase as a. Description genetic reporters are used commonly in cell biology to study gene expression and other cellular events coupled to gene. Coelenterazine is an imidazolopyrazine derivative which, when oxidized. Because of the requirement of atp, firefly luciferases have been used extensively in biotechnology.
The gene encoding renilla luciferase is fused to a constitutive promoter pgk1 or spt15 and integrated into the yeast genome at the can1 locus as a control. Less than 10 20 moles of luciferase have been detected under optimal conditions. It is widely used because it is convenient, relatively inexpensive, and gives quantitative measurements instantaneously. Luciferase as a reporter of gene activity in plants. Firefly luciferase is the ideal reporter gene for monitoring circadian rhythms in eukaryotes. A codon optimized and stabilized luciferase gene based upon the sequence of the natural luciferase gene isolated from luciola cruciata japanese firefly and a novel recombinant dna characterized by incorporating this new gene coding for a novel luciferase into a vector dna for improved activities in mammalian cells, are disclosed. The luciferaseluciferin noninvasive bioluminescent reporter genereporter probe imaging system. A luciferase assay is used to determine if a protein can activate or repress the expression of a target gene. Pdf nanoluc reporter for dual luciferase imaging in.
Luciferase based reporter gene assays are widely used because of their ultrasensitive detection capacity and wide dynamic range. The hrpconjugated antirabbit igg antibody gtx211001 was used to detect the primary antibody. The oneglo luciferase assay buffer and oneglo luciferase assay substrate, provided with this system, are combined to form the oneglo reagent see section 3. Expression of the luciferase gene fusion product confers on the host the ability to. Download the latest version of genetexs pamphlet for western blot troubleshooting.