...uses for plasmids expressing luciferase Luciferase reporter assay The typical luciferase-containing plasmid...promoter linked to luciferase. Activation of the pathway will result in luciferase expression and more complex...cloning of firefly luciferase was published in 1985, several studies utilized luciferase as a genetic reporter...mammalian cells. Luciferase assays have since become a gold standard in gene expression analysis and a ...Well-established Biological sources of luciferase Although luciferase isolated from the firefly beetle (Photinus...second most commonly used luciferase. Other organisms from which luciferases have been cloned from include...gene expression. The regulatory element (wild-type or mutant) is cloned upstream of the luciferase gene...

...codon-optimized Firefly luciferase genes (e.g., luc2), and NanoLuc® Luciferase are available from the ... creation of NanoLuc® Luciferase from a rather dull 19kDa Oplophorus luciferase by Promega’s Advanced ...than our more common luciferases like firefly (FLuc) and Renilla (RLuc) luciferase. This is important not...coupled luciferase. This strategy was first applied using RLuc8, an engineered Renilla luciferase, to produce...isn’t science better when we share? NanoLuc® Luciferase-based tools are fantastic examples of technologies...bioluminescence to overcome these issues as traditional luciferases emit at near optimal wavelengths (Fluc emits ...sources. Unfortunately, detecting traditional luciferases’ dim light emissions requires long acquisition...

...Administration of Substrate: Some luciferase reporters, such as Renilla luciferase (RLuc) or NanoLuc, require...bacterial reporters that use fluorophores and luciferases to track bacteria in real time, but each of these...Bourdeau et al created two constructs optimized for expression in E. coli: acoustic gene 1 (arg1) and acoustic... growth following acoustic collapse E. coli re-express arg1 gas vesicles and restore their ultrasound ...for ARG’s: in vivo imaging. Specifically, arg1 expressing E. coli aided in ultrasound imaging of the mouse... bowel conditions. In these experiments, arg1 expressing ECN were injected into the mouse colon, imaged...the images compared to mice injected with ECN expressing lux. arg1 and lux reporters both allow for visualization...

...Renilla luciferase variant fused to an FP; when supplied with a luciferase substrate, the luciferase transfers...By itself, Renilla luciferase has a low quantum yield, and when transiently expressed in cells, generates...Nano-lantern, a fusion of a mutagenized Renilla luciferase (RLuc8) with Venus. This luminescent technology...transfer (BRET). Photons emitted by a Renilla luciferase variant are used to excite the fused fluorescent...need for an excitation light source. Instead, a luciferase substrate like coelenterazine is supplied in ...eNL constructs use the brightest characterized luciferase, NanoLuc, and its substrate furimazine. In addition... luciferase...

...bacterial expression vector which gives close to 100% cloning efficiency and gene expression. The pUCXKT... SFFV) or inducible (CMV-TO) cDNA expression, sgRNA (U6) expression, shRNA/shRNA-miR30 constitutive (H1...vector expressing a separate reporter gene (EGFP, iRFP, IFP1.4, puromycin, neomycin or luciferase) to create...enhancing target gene expression anywhere from 87x to 20,000x over endogenous expression (depending on the...their appropriate functions affecting luciferase reporter expression as anticipated. For instance, an aptamer...activated gene expression from the reporter construct. The sgRNA targeting Gaussia luciferase can be easily...Testing your cell-free expression system: There’s a kit for that! Cell-free expression systems have been ...

...When this happens, BiT and the LgBiT luciferase fragment (expressed separately) assemble and catalyze the...difficult to study in vitro, requiring either expressing it with additional cofactors or using significantly...BY license. This construct can be easily expressed and purified from E. coli, retains important functional...elegans and included artificial introns to improve expression. When using these constructs to tag the GOLG-...photostability and also matured considerably faster, with expression levels comparable to GFP during early embryogenesis... tissues like the intestine. Figure 3: Expression of fluorophore-tagged GOLG-4 through eight stages...applications needing intense brightness (such as low-expression proteins of interest) and mScarlet-I3 for scenarios...

... the split-luciferase system, intein-mediated splicing reconstitutes functional luciferase only when fusions...activation. Vary the expression levels of bait and prey proteins - overexpression can lead to false interactions...During this period, proteins were successfully expressed as fusions that retain their individual activities...target gene, in this case a reporter gene (e.g. luciferase or GFP), regardless of whether it interacts with...see Figure 2a) (4). These fusions are then co-expressed in a yeast strain that also contains a reporter...bait”, Figure 2b) cannot activate reporter gene expression on its own, but, if the two POI’s interact, this...the promoter of the reporter gene to activate expression (Figure 2c). The reporter gene then allows detection...

...viral infection), producing firefly luciferase. Firefly luciferase can readily be measured to quantify...single (non-viral) reporter gene (e.g. firefly luciferase or eGFP) flanked by the EBOV-specific gene start...plasmids allows researchers to partially control expression levels of each gene by altering the amount transfected...to transcribe the reporter gene (e.g. firefly luciferase). The polymerase recognizes the gene start signal...RNA polymerase gene or using cells that stably express T7 (e.g. BSR T7/5 cells) (1). We hope that minigenomes...

...observe expression from Agrobacterium, but not the plant. For example, we have seen strong expression of the... transgenic cultures or plants, our transient expression method enables us to quickly screen and investigate...reporter gene so that the transformation and gene expression can be tracked. During a transient leaf transformation...Once we were on the right track, we switched to luciferases to more easily quantify the effect of the different...GUS::tMAS to confirm transformation success. Luciferases are great quantitative reporters and use a substrate...substrate to produce bioluminescence. Luciferases usually have shorter mRNA and protein half-lives then...References D'Aoust, Marc-André, et al. "Transient expression of antibodies in plants using syringe agroinfiltration...

...determined by comparing cells expressing only eGFP expression to cells expressing eGFP and mCherry. ACE was...mCherry and a T2A site and expressed in cells. mCherry is constitutively expressed to identify successfully...that can be repaired by NHEJ restoring mCherry expression. Cells can then be sorted using Fluorescence-...Reporters in Gene On include a mScarlet reporter, luciferase reporter, and neomycin resistance reporter. Image...integrated it into human and mouse cells that expressed either Cas9 or an optimized cytidine base editor... of different reporters including mScarlet-I, luciferase, antibiotic resistance markers, and other enzymes... of a mutated start codon to initiate protein expression, it can be used for an array of detectable proteins...

...could enter cultured cells and activate expression of a luciferase reporter. CPP was not required for protein... mutations and gene expression changes elsewhere in the genome. Second, expression is by nature transient...cells! After verifying the specificity of their luciferase assay, they concluded that components in the ...stem cells transduced with iTOP resulting in GFP expression. Image courtesy of Diego D'Astolfo. CRISPR...causes a cell to take up, and continued protein expression would require multiple transduction events. For... to binary systems in which transient protein expression has a long-lasting effect, such as Cre- or Cas9...

...mouse retina. GFP expression robustly activated the reporter gene TdTomato, whose expression was absent without...reporter expression was induced in ~76% of GFP+ cells, and 100% of DsRed+ cells also expressed GFP. Using.... Cepko lab researcher Jonathan Tang wanted to express a variety of proteins in single cell types in the... could use previously established mouse lines expressing GFP in specific cell types to do more than just...cells. If GFP could be co-opted to control gene expression, he could then selectively manipulate only GFP-labeled...GBPb-GAL4 (DNA-binding domain); and a UAS-driven luciferase reporter construct. In the absence of GFP, no...create a complete transcription factor, activating luciferase transcription. Tang et al. coined this system...

...protein is localized, and how its localization and expression may change across various conditions. However...perfect. For example, If your fusion protein is expressed at low levels, you have to increase your imaging... fused to the cognate scFV antibody, which is expressed from a separate plasmid. This system amplifies.... then tested their hypothesis that the lower expression levels of SunTag constructs would avoid negative...organelle toxicity. First generation (v1) SunTag is expressed at very low levels due to poor stability of the...the GCN4 scaffold. To increase expression, Tanenbaum et al. modified the GCN4 sequence to increase its...protein-tagging system for signal amplification in gene expression and fluorescence imaging.” Cell 159(3) (2014)...

...Vital component for expression vectors: determines which cell types the gene is expressed in and amount of...sites allowing for the easy insertion of DNA. In expression plasmids, the MCS is often downstream from a ...Generally, scientists use plasmids to manipulate gene expression in target cells. Characteristics such as flexibility...common plasmid types include cloning plasmids, expression plasmids, gene knock-down plasmids, reporter ...Green Fluorescent Protein (GFP) Plasmids 101: Luciferase Monitor the level of a chemical in a particular...

...light include chemiluminescent proteins, like luciferases, as well as fluorescent ones, like Green Fluorescent...in 1994, Prasher’s collaborator Martin Chalfie expressed GFP in exogenous organisms (E. coli, and later...

...pseudoviruses express a luciferase reporter which is used as a readout for infection of ACE2-expressing target...spike variants. “We created a series of spike expressing plasmids which encode spikes found on all of ...

... HEK293FT cells expressing a single gRNA targeting a single gene. The gRNA expressing cells were then ... to test multiple expression systems before they achieve the appropriate expression level for their gene...create protein expression constructs fused with epitopes or fluorophores, generate expression vectors with... lab’s own pAc-sgRNA-Cas9 insect expression backbone, which expresses the gRNA from a Drosophila U6:2 ...Protein A-Renilla luciferase and the “prey” proteins are tagged with Firefly luciferase. The bait and prey...model organisms. As long as it was expressed in a cell that also expressed auxin-sensitive TIR1, any protein...cell lines expressing it. The Church lab fixed this issue by creating a new dCas9-VPR expression plasmid ...

... ligands remain unknown. Together, NRs govern expression of genes involved in a broad range of reproductive...efficient and timely activation or repression of gene expression (see www.nursa.org for a curated list of coregulators...ligand, cell type, and promoter, organizing the expression of functional groups of genes involved in the...Our Q-PCR datasets, for example, document the expression patterns of nuclear receptors across multiple...possible without the development of a vast number expression and reporter plasmids and the free exchange of...evolved from chloramphenicol acetyltransferase to luciferase, the basic assay remains widely used today.  ..., B. W. (2002). Combinatorial control of gene expression by nuclear receptors and coregulators. Cell 108...

...biosensor system was developed by constructing 14 luciferase donor Rluc8-Gα chimeras, four acceptor GFP2-Gγ...TRUPATH system, cells are transfected with plasmids expressing the GPCR of interest, a Gα-Rluc8 plasmid, and...

...type 2 (Th2) cells from mice that constitutively express Cas9 and screen for genes that affect Th2 differentiation...differentiation and activation, using expression of differentiation factors as the screen readout. This retroviral...ApoB-NanoLuc reporter by inserting the NanoLuc luciferase downstream of the apoB gene. Using this reporter...

... yeast expression, worm expression, insect expression, plant expression, mouse targeting, CRISPR, TALEN... mammalian expression, bacterial expression, lentiviral, retroviral, AAV, RNAi, luciferase, cre/lox, yeast...

...Description Expression Additional considerations CMV General expression mRNA Strong mammalian expression promoter...lower expression than U6. May have better expression in neuronal cells. U6 small RNA expression shRNA...some cell types. EF1a General expression mRNA Strong mammalian expression from human elongation factor ...give consistent expression regardless of cell type or physiology. SV40 General expression mRNA Mammalian...  Commonly used in expression systems for Drosophila. Polyhedrin General expression mRNA  Strong insect...Constitutive  Commonly used in expression systems for insect cells. CaMKIIa Gene expression for optogenetics mRNA...neuronal/CNS expression. Modulated by calcium and calmodulin. GAL1, 10 General expression mRNA Yeast ...

...the receptor, which can then activate expression of a luciferase reporter in the nucleus. The Roth lab...resource called PRESTO-TANGO (parallel receptorome expression and screening via transcriptional output - TANGO...preps from DREADD plasmids allow you to easily express a DREADD receptor in a specific neuronal subtype...

... for each gene that is expressed, multicistronic vectors simultaneously express two or more separate proteins...cells that are expressing your gene of interest at high levels. Unlike vectors that express screenable or...want to express multiple genes in the same cell. Although it is possible to drive co-expression by using... with multiple, individual expression cassettes, having the genes expressed from the same cassette is ...designed for bicistronic expression; however, many could be easily manipulated to express more than two genes...Co-expression of multiple genes is valuable in many experimental settings. To achieve this, scientists...multicistronic vectors? Detecting cells that are expressing your gene, especially if you are studying a novel...

...Knockdown the expression of Gene X using shRNA expressed from a plasmid   Figure 1: Expression Level of Gene...as the expression level of Gene X in cells treated with Plasmid A is similar to the expression level of...plasmid that constitutively expresses a reporter protein (e.g., GFP or luciferase) that is either co-transfected...decreased expression of Gene X. Ergo the positive control(s) should decrease the expression of Gene X....into the cells and the ability of the cells to express the protein.   In analysis of the result in Figure...Figure 1, an internal control, such as the GFP-expressing Plasmid B, could demonstrate whether the cells...cells were transfected successfully and expressing the protein. For example, fluorescence microscopy images...

...accepts the sgRNA target sequence for U6 promoter expression and the second site accepts the homology arm ...-injection of the targeting vector and a Cas9 expression plasmid will insert the desired genetic tag and...donor plasmids for protein tagging, including luciferase (NanoLuc), fluorescent proteins (TagGFP2, TagBFP... then mixed and transfected along with a Cas9 expression vector. They can also be introduced directly ...directly into a Drosophila S2 cell line stably expressing Cas9. This PCR toolkit offers C- and N-terminal tagging... template for inducible alteration of protein expression levels. CRISPR tagging systems offer users an... tag endogenous proteins for studying protein expression, localization, and protein-protein interactions...

...same strain. MazF expression is under control of the AOX1 promoter whose expression is induced by methanol...that are used to drive high protein expression in vivo. Expression plasmids are often lost without plasmid... and James B. Kaper. "Use of stabilized luciferase-expressing plasmids to examine in vivo-induced promoters...hok/sok system to create strains that can stably express antigens in sufficient quantities to generate an...plasmid R1: Sok antisense RNA regulates hok gene expression indirectly through the overlapping mok gene."...

...cell-derived neurons expressing ASAP5-Kv. C) Cultured rat hippocampal neuron expressing pan-membrane ASAP5...Figure 2), the authors developed a cost-effective expression and labeling approach using these constructs ...protein of interest can be inserted. B) Protein expression and labeling procedure. Adapted from Smith et..., and CLAP-tagged proteins for mammalian cell expression. These plasmids will be useful not only for future...Janicot and Maziarz et al., 2024). Each plasmid expresses all the components of the biosensor in optimal... a YFP-tagged G protein and a NanoLuc® (Nluc) Luciferase-fused detector that specifically binds to active...

...bacterial genome, and inserted it into E. coli to express and then test for any proteins that could cut RNA..., when they looked at the transformed E. coli expressing Cas7-11, they found no evidence of growth inhibition...RNA editing tools.   Fig. 1 Schematic of luciferase RNA knockdown assays using Cas7-11   Use ...

...binding sites increased activation of HIV promoter-luciferase constructs. In multiple HIV-1 latent T cell lines...HIV-1, measured via HIV-1 promoter-driven GFP expression. In cell lines that can produce HIV-1 toxic proteins... gRNAs to target the HIV-1 5’ and 3’ LTRs and expressed them along with Cas9 in the T-cell line 2D10. ...of the LTRs joined together. Clonal Cas9/gRNA-expressing cell populations were also immune to HIV-1 reinfection...cycle progression, or apoptosis with Cas9/gRNA expression. A pooled analysis found no evidence of Cas9 .... For two HIV-1 strains, Cas9/gRNA lentiviral expression significantly reduced the HIV-1 copy number, ...preventing re-infection would require sustained expression of Cas9/gRNAs, which could result in higher rates...