... Figure 4: Reporters in Gene On include a mScarlet reporter, luciferase reporter, and neomycin resistance...without relying solely on GFP (Katti et al., 2020). They named this reporter “Gene On” or GO. GO works by...mediated editing. eGFP reporters for single base editing by APOBEC-Cas9 Though the ACE reporter quantifies APOBEC-Cas9...DSB in their previous reporter, the Harris Lab created a panel of eGFP reporters to quantify the on-target...editing reporters. Image from St. Martin et al., 2019. The Harris lab used these eGFP reporters to analyze... their hypothesis with GFP fluorescence. To do this they generated a silent GFP construct, that contains... of eGFP reporters for single base editing by APOBEC-Cas9 editosome complexes. Scientific Reports 9: ....

...following components: a GFP protein that binds to the coding region of a reporter mRNA via a PP7 coat protein...protein (NLS-PCP-GFP) an RFP protein that binds to the 3’ UTR of the reporter mRNA via an MS2 coat protein...64543) Before translation, both GFP and RFP are bound to the reporter, causing it to appear yellow. The...translation pushes NLS-PCP-GFP off of the mRNA, leaving only a red FP bound to the reporter. Because it contains...translation is necessary for removal of GFP from the TRICK reporter. Delayed translation is known to be important... Drosophila patterning gene oskar. Early in development, the osk-TRICK reporter was double labeled, indicating...correlated with GFP intensity, showing that TRICK is an accurate readout of relative reporter translation....

...GBPs/GFP. The new system, CRE-DOG (Cre Dependent On GFP), is activated by GFP and derivatives GFP and ... of GFP. These systems, known as T-DDOG and Cre-DOG, respectively, repurpose popular GFP reporter lines...to bind GFP. The idea of using GFP as a scaffold suddenly seemed very realistic. Envisioning GFP as a substrate...a UAS-driven luciferase reporter construct. In the absence of GFP, no reporter output was observed in ...electroporated T-DDOGs, GFP, and a reporter construct into the mouse retina. GFP expression robustly activated...activated the reporter gene TdTomato, whose expression was absent without electroporated GFP. They then ...T-DDOGs with two mouse GFP reporter lines; again, TdTomato was seen only in cells with GFP, at a high activation...

...apoptosis reporter, ZipGFP, which utilized the self-assembling split GFP (GFP1-10 and GFP11) to increase...self-assembling split GFP (GFP1-9 and GFP10-11) by flipping a single β-strand of GFP, hence the name FlipGFP. FlipGFP... used genetically encoded fluorescent executioner caspase reporters. Several apoptosis reporters have ...fold compared to other reporters (Zhang et al., 2019). How does FlipGFP work? GFP contains 11 β-strands...transfer (FRET)-based executioner caspase reporters. In these reporters, the FRET pair is linked by a caspase...FlipCherry: Red fluorescence protease reporter To add to the fluorogenic reporter toolkit, the Shu Lab also designed...sensor based on tripartite split-GFP association." Scientific reports 3 (2013): 2854. PubMed PMID: 24092409...

...detectable by two different tags: a reporter and an HA tag. The reporter comes in three varieties: HRP, Spaghetti...independent of the reporter. The only caveat is that HA staining is weaker than reporter staining. Picking...Picking the right reporter for your RANBody While all three reporters stain cultured cells and tissue sections... have 10 HA or 10 MYC tags built into a GFP scaffold. Some GFP nanobodies  stain poorly when tagged with...Monster, and Chicken IgY Domain RANbody Reporters. Reporter Detection Method Uses Pros Cons Antigens...weak signals of fluorescent protein (i.e. GFP, RFP) Detecting GFP reconstitution across synaptic partners...recombinant DNA, they can’t be genetically tagged and instead are conjugated to reporters (i.e. fluorescent dyes...

... to stably integrate a reporter gene that contains a puromycin resistance gene followed by the target ...histone)-GFP reporter into the genome of mammalian cells.  Figure 1: The SRIRACCHA reporter construct...that targets your desired gene (whose sequence is also found in the reporter gene). A control RFP expression...Find GEmCherry2 reporter plasmids at Addgene Overall SRIRACHA and GEmCherry2 reporter systems provide ... CRISPR reporters in the near future.  SRIRACCHA (stable, but reversible, integrated reporter for assaying...that contains a puromycin resistance gene linked to an in-frame H2B-GFP. This visual read out allows for ...will express RFP while cells containing the reporter gene that was cleaved by Cas9 and repaired via HDR...

...scientists to begin to study GFP and deduce its biochemical properties. Now GFP and its fluorescent derivatives...and scientists employ GFP for a wide number of functions, including: tagging genes for elucidating their...constructing fluorescent fusions. Transcription Reporter: Placing GFP under the control of a promoter of interest...monitor gene expression from that promoter in a given cell type. This type of transcription reporting was ...type may have the GFP under the control of an additional promoter from that of the gene of interest, or ...expressing GFP from cells that are not. Developmental/transgenic uses: Because of its stability, GFP can be...implantation and generation of transgenic mice. Purification: GFP can be used as a general epitope tag for...

...decrease in red puncta. Find the mRFP-GFP-LC3 plasmid at Addgene! GFP-LC3-RFP-LC3∆G: A fluorescent probe ...flux. mRFP-GFP-LC3 functions as an autophagy bionsensor via the same mechanism as Rosella: GFP is quenched...autophagy biosensor is the GFP-LC3-RFP-LC3ΔG probe. It’s similar to the mRFP-GFP-LC3 Tandem Fluorescent Protein...Autophagic flux is estimated by the GFP/RFP ratio: a decrease in GFP fluorescence relative to RFP fluorescence...the mitochondria-targeted Keima variant at Addgene! mRFP-GFP-LC3 tandem fluorescent protein This autophagy...translation, this fusion protein is cleaved to generate one molecule of GFP-tagged LC3 for every one molecule of...decrease in autophagic flux.   Find the GFP-LC3-RFP-LC3∆G plasmid at Addgene! References Klionsky, Daniel J....

...study the A. victoria gene responsible for encoding GFP, and, in 1992, he reported its sequence. Soon after...laboratory. The discovery of GFP The story of how fluorescent proteins like GFP came to be used as research...their work on GFP. This great honor serves to highlight how useful fluorescent proteins like GFP really are...fluorescent ones, like Green Fluorescent Protein (GFP). These molecules occur naturally in bioluminescent... calcium ions, and the second, which we now call GFP, absorbs that blue light and glows green. In the ...new green glowing protein could be used to measure gene transcription, as well as to track protein localization... Prasher’s collaborator Martin Chalfie expressed GFP in exogenous organisms (E. coli, and later C. elegans...

...remove a stop cassette upstream of an effector/reporter gene. In cells where the stop cassette is removed...https://doi.org/10.1101/788679 A destabilized GFP reporter for calcineurin activity in yeast Article contributed...that target calcineurin. Current calcineurin gene reporters are based on the bacterial βgal assay, and ...calcineurin transcriptional reporter consisting of a quickly-degraded destabilized GFP variant fused to a 4X...bottom panels) equipped with destabilized GFP calcineurin reporter (blue) and stained with PI (pink) to indicate... Image from Diessl et al., 2020. Find the GFP reporter calcineurin activity here Diessl et al., Microbial...the sgRNAiBAR library covers every annotated human gene, it enables genome-wide screening with a high MOI...

...sensor was engineered from a GFP variant and a DA receptor such that GFP fluoresces when the receptor ...on a similar strategy: a GFP variant bound to a DA receptor, such that the GFP fluoresces upon DA-receptor...Record DA dynamics of optogenetically activated and inhibited neurons in live mice Report modulation of DA ...behaving mice to gather the following types of data: Report time-course and concentration of released DA in...moving mice to gather the following types of data: Report endogenous DA release from a single dopaminergic...looming stimulus in vivo in transgenic zebrafish Report DA release in live mice over the course of Pavlovian...viral vectors 101 eBook Resources on Addgene.org Learn about Addgene's viral service Find viral protocols...

...line expressing a GFP-tagged version of a protein of interest. The binding of the GFP nanobody to its target... proximity to the GFP-tagged protein (albeit slightly offset due to the nanobody-GFP interaction). The...-SSAP) which insert genes in a cleavage free manner. The dCas9-SSAP system reports an efficiency of up...ligase TurboID fused to a conditionally stabilized GFP-nanobody rather than a specific protein of interest... For a one kilobase insertion, the researchers reported an editing error rate of less than 0.3%, both ...Fig. 3: Development of a cleavage-free dCas9-based gene editor using microbial SSAPs. Image from Wang, et...using mStrawberry, localization peptides, and reporters that drive the expression of the fluorescent protein...

...Green Fluorescent Protein (GFP/EGFP) for a transcriptional reporter or as a general cytoplasmic label of cells...remain unaware that EGFP/GFP is prone to forming noncovalent dimers. This property of EGFP can lead to significant...contributed by guest blogger Erik L. Snapp. Stop using EGFP/GFP for fusion proteins! Despite multiple studies ...in the world, is not inert. In all seriousness, EGFP/GFP has a real nontrivial propensity to noncovalently...best solution is to use truly monomeric FPs. EGFP and other GFP family members (superfolder, Emerald, CFP...However, if you fuse your protein of interest (POI) to GFP to study the protein's behavior in cells, in solution...noncovalently dimerize. That means that your POI fused to GFP or another fluorescent protein (FP) could be forming...

...to further restrict gene activation to cells expressing both Cre and GFP. The GFP dependent FLPo, Flp-...respectively of a GFP dependent Cre. The two segments come together in the presence of GFP to form a functional...extensive supply of transgenic reporter lines that supply a variety of GFP expression patterns, thus allowing...AAVrg pAAV-hDlx-Flex-GFP-Fishell_6 Gordon Fishell   Optogenetics Optogenetics continues to supply neuroscience...-2 proteins ChR2 for targeted expression DREADDs GFP dependent Cre and FLPo And more!  New lentiviral...inhibition library that inhibits over 18,000 human genes and is used for genome-wide inhibition screening...in new serotypes, as well as a new Cre dependent GFP control plasmid for expression in forebrain GABA-...

...dim GFPs The availability of the three dimensional structure of GFP, its brighter mutant S65T-GFP, and...and GFP shRNA. Mice carrying the R26DsRedR; CRUSH-GFP; and Nestin-Cre alleles showed efficient GFP knockdown...be used to generate random mutagenesis libraries or to insert heterologous genes, reporters, or other ...they can successfully create GFP fusion proteins with a variety of genes across the genome. Although this...effectively targets your gene/region of interest is a key step for CRISPR/Cas9 gene editing. Dr. Masahito...Masahito Ikawa has created a GFP reporter plasmid for scientists to validate the efficacy of their gRNAs. The...sequence or gene of interest into the pCoofy vectors, select the appropriate pair of vector and gene primers...

...widely used luciferase reporter gene plasmid pGL3 pGL3 luciferase reporter gene plasmids have been used... cassette to the gene of interest. The gene to be expressed is amplified using a gene-specific forward...destination vector expressing a separate reporter gene (EGFP, iRFP, IFP1.4, puromycin, neomycin or luciferase...sgRNA-targeted gene, resulting in increased activation of endogenous transcription of the target gene. The brilliant...of downstream genes. Nihongaki et al describe the targeted activation of endogenous genes and detail the...efficiency and gene expression. The pUCXKT vector ensures that all screened plasmids contain a gene variant*...resistance gene (missing the first two codons). Two stop codons are inserted between the truncated gene and ...

...S65T) in the original jellyfish GFP (AvGFP) to create enhanced GFP (EGFP), a variant with improved brightness... There are numerous GFP variants. Here I use the name ‘GFP’, which refers to mEGFP. This variant was made...examined on a case-by-case basis. GFP is sensitive to acid The chromophore of GFP can exist in a protonated and...its acid tolerance is maintained in cells.   GFP is big GFP is a 28 kDa protein that resembles a cylinder...2013; Hilaire et al., 2017). GFP can only be used for tagging proteins GFP fusion proteins are made by ... a fusion with GFP allows for detection of genomic locations (Chen et al., 2013). GFP fluorescence overlaps...necessary to generate the strictly monomeric EGFP variant, mEGFP (Zacharias et al., 2002). GFP needs oxygen...

...process worked well with the common GFP variants EGFP, mGFP4-T, and S65T GFP, and no red color was observed...repurpose GFP is great news for microscopy enthusiasts! Addgene has empty backbones containing GFP variants...you’ve used a GFP derivative to conduct subcellular localization studies or make a reporter construct. Fluorescent...exciting GFP-labeled plant cells at 405 nm instead of 488 nm, Sattarzadeh et al. noticed that GFP had turned...cytosol-targeted GFP. White circles represent the irradiated area. Photoconverted GFP spreads through ...Applications and limitations of GFP photoconversion One concern with photoconverting GFP is the expression level...intensity increases with GFP concentration and saturates around 30 uM. For low-abundance GFP fusions, a photoconversion...

...fundamentally the reporter construct monitors the expression of a nearby gene. The reporter gene serves as a...exon of a gene, and thus they do not disrupt the gene expression of the gene they monitor. Gene traps Gene...the gene of interest in any way. For example, if green fluorescent protein (GFP) was the reporter gene...stimulate the expression of the reporter. The system is made up of a reporter gene flanked by a minimal promoter...a promoter-less reporter which needs to integrate within an exon of a gene to generate a transcriptional...predicted genes have unidentified roles as well (Vanni, et al). Is it true that this many genes are truly...? Traps – gene, enhancer, and promoter – all rely on the random integration of a reporter construct in...

...Fluorecent Proteins in Nano Lanterns Reporter gene assays A reporter gene, such as luciferase, usually serves...since become a gold standard in gene expression analysis and a luciferase gene (one of many available to choose...enhancer regions on gene expression can be determined by detection of the reporter in a specific assay...excite GFP. As a result of the different mechanisms to produce photons, chemiluminescent reporters are generally...bacterial cells. Expression of luciferase as the reporter gene is measured via its activity to produce light..., several studies utilized luciferase as a genetic reporter in plant and mammalian cells. Luciferase assays... is also the most commonly used bioluminescent reporter. This beetle emits a yellow-green light with a...